阅读说明：本技术 用于生物标志物分析的设备、系统和方法 (Devices, systems, and methods for biomarker analysis ) 是由 迪尔克·万登博姆 马蒂亚斯·埃里克 保罗·厄特 吉姆·肖瓦普恩 于 2018-09-26 设计创作，主要内容包括：本文提供了用于使用少量母体生物样品中的无细胞胎儿核酸预测或确定胎儿性别的设备、系统、试剂盒和方法。设备可以在妊娠早期在需求点处使用,并且与通信设备兼容。(Provided herein are devices, systems, kits and methods for predicting or determining fetal gender using cell-free fetal nucleic acid in a small maternal biological sample. The device may be used at a point of need early in pregnancy and is compatible with communication devices.)

1. An apparatus, comprising:

a) a sample purifier for removing cells from a biological fluid sample to produce a cell-removed sample; and

b) at least one of a detection reagent and a signal detector to detect a plurality of cell-free DNA fragments in the cell-depleted sample.

2. The apparatus of claim 1, wherein a first sequence is present on a first cell-free DNA segment of the plurality of cell-free DNA segments and a second sequence is present on a second cell-free DNA segment of the plurality of cell-free DNA segments, and wherein the first sequence is at least 80% identical to the second sequence.

3. The apparatus of claim 2, wherein the apparatus comprises at least one nucleic acid amplification reagent and a single pair of primers capable of amplifying the first sequence and the second sequence.

4. The apparatus of claim 2, wherein at least one of the first sequence and the second sequence is repeated at least twice in the genome of the subject.

5. The apparatus of claim 2, wherein the first sequence and the second sequence are each at least 10 nucleotides in length.

6. The apparatus of claim 2, wherein the first sequence is on a first chromosome and the second sequence is on a second chromosome.

7. The apparatus of claim 2, wherein the first sequence and the second sequence are on the same chromosome, but separated by at least 1 nucleotide.

8. The apparatus of claim 2, wherein the first sequence and the second sequence are functionally connected.

9. The apparatus of claim 1, wherein the sample purifier comprises a filter, and wherein the filter has a pore size of about 0.05 microns to about 2 microns.

10. The apparatus of claim 9, wherein the filter is a vertical filter.

11. The apparatus of claim 1, wherein the sample purifier comprises a binding moiety selected from the group consisting of an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, and combinations thereof.

12. The device of claim 11, wherein the binding moiety is capable of binding to an extracellular vesicle.

13. The apparatus of claim 2, wherein the at least one nucleic acid amplification reagent comprises an isothermal amplification reagent.

14. The device of claim 1, wherein the signal detector is a lateral flow strip.

15. The apparatus of claim 1, wherein the apparatus is contained in a single housing.

16. The apparatus of claim 1, wherein the apparatus operates at room temperature.

17. The apparatus of claim 1, wherein the apparatus is capable of detecting a plurality of biomarkers in the cell removal sample within about five minutes to about twenty minutes of receiving the biological fluid.

18. The apparatus of claim 1, comprising a communication connection.

19. The apparatus of claim 1, comprising a transcutaneous puncturing device.

20. A method, comprising:

a) obtaining a fluid sample from a subject, wherein the volume of the biological sample is no greater than about 120 microliters;

b) contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sequence of interest to produce an amplification product; and

c) detecting the presence or absence of the amplification product, wherein the presence or absence is indicative of a health state of the subject.

21. The method of claim 20, wherein the fluid sample is a blood sample.

22. The method of claim 20, wherein the fluid sample is a plasma sample from blood.

23. The method of claim 22, wherein the plasma sample has a volume of no more than 50 μ l.

24. The method of claim 22, wherein the volume of the plasma sample is from about 10 μ l to about 40 μ l.

25. The method of claim 20, wherein the sample contains about 25pg to about 250pg of total circulating cell-free DNA.

26. The method of claim 20, wherein the sample contains from about 5 to about 100 copies of the sequence of interest.

27. The method of claim 26, wherein the copies are at least 90% identical to each other.

28. The method of claim 20, wherein the sequence of interest is at least 10 nucleotides in length.

29. The method of claim 20, wherein contacting comprises performing isothermal amplification.

30. The method of claim 20, wherein contacting occurs at room temperature.

31. The method of claim 20, wherein the method comprises incorporating a tag into the amplification product while the amplification occurs, and wherein detecting the presence of the amplification product comprises detecting the tag.

32. The method of claim 31, wherein the tag does not comprise a nucleotide.

33. The method of claim 31, wherein detecting the amplification product comprises contacting the amplification product with a binding moiety capable of interacting with the tag.

34. The method of claim 33, comprising contacting the amplification product with the binding moiety on a lateral flow device.

35. The method of claim 20, wherein steps (a) through (c) are performed in less than fifteen minutes.

36. The method of claim 20, wherein the method is performed by the subject.

37. The method of claim 20, wherein the method is performed by an individual who has not received technical training for performing the method.

38. The method of claim 20, wherein obtaining, contacting, and detecting are performed using a single handheld device.

39. The method of claim 20, wherein the health state is selected from the presence or absence of pregnancy.

40. The method of claim 20, wherein the health state is selected from the group consisting of the presence or absence of a neurological disorder, a metabolic disorder, cancer, an autoimmune disorder, allergy, and infection.

41. The method of claim 20, wherein the health state is a response to a drug or therapy.

42. An apparatus, comprising:

a) a sample purifier that removes cells from a fluid sample of a female subject;

b) at least one nucleic acid amplification reagent;

c) at least one oligonucleotide comprising a sequence corresponding to the Y chromosome, wherein the at least one oligonucleotide and nucleic acid amplification reagents are capable of producing an amplification product; and

d) at least one of a detection reagent or a signal detector for detecting the amplification product.

43. The device of claim 11, wherein the oligonucleotide comprises a sequence corresponding to a gene selected from the group consisting of DYS14 or TTTY 22.

44. A method, comprising:

a) obtaining a fluid sample from a female pregnant subject, wherein the volume of the biological sample is no greater than about 300 microliters;

b) contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sex chromosome; and

c) detecting the presence or absence of amplification products, wherein the presence or absence is indicative of the gender of the fetus of the female pregnant subject.

45. The method of claim 43, wherein the fluid sample is a blood sample.

46. The method of claim 44, wherein the volume of the blood sample is no greater than 120 μ l.

47. The method of claim 43, wherein the fluid sample is a plasma sample from blood.

48. The method of claim 46, wherein the volume of the plasma sample is no greater than 50 μ l.

49. The method of claim 46, wherein the volume of the plasma sample is from about 10 μ l to about 40 μ l.

50. The method of any one of claims 43-48, wherein obtaining comprises performing a finger prick.

Background

Genetic testing is a means of obtaining information about the subject's DNA and/or expression of that DNA. Genetic testing is constantly being developed to obtain biological information about a subject. The biological information has many uses, including determining the health status of an individual, diagnosing an individual as having an infection or disease, determining a suitable treatment for an individual, addressing criminal issues, and determining paternity. Currently, genetic testing is performed in clinics and laboratories primarily by trained personnel using expensive and bulky equipment that requires technical training and expertise to use. From the time a biological sample is obtained from a patient, it typically takes several days to weeks to provide the patient with the results of the genetic test.

For example, many people who are aware of pregnancy desire to know the gender of the child (referred to throughout this application as gender) as soon as possible. There are several tests that allow gender information to be obtained from DNA in maternal blood. The blood obtained from the mother must be analyzed by a trained technician using sophisticated equipment. If the blood is obtained at a location remote from the laboratory where the DNA analysis is performed, the sample must be stored, shipped and analyzed in a timely manner, otherwise there is a risk of sample degradation.

Disclosure of Invention

Disclosed herein are devices, systems, kits, and methods for analyzing components (e.g., nucleic acids, proteins) of biological samples, including samples from animals (human or non-human), environments (e.g., water, soil), plants, bacteria, and food. In general, the devices, systems, kits, and methods disclosed herein are capable of providing genetic information from very low volume samples by utilizing cell-free DNA fragmentation. Cell-free DNA fragmentation produces statistically independent markers from repetitive regions (e.g., regions with a common sequence) and/or multiple detection regions along a target region. By way of non-limiting example, cell-free DNA fragments from repetitive regions (e.g., genomic regions containing multiple copies of the same or similar sequences) are present at a higher effective concentration in a sample relative to DNA fragments having sequences that are not present in multiple copies. Advantageously, fragments from the repeat region can be amplified with a single primer pair or detected with a single probe. However, the multiple detection regions do not necessarily share similar sequences. Such fragments can also be detected in small volumes, e.g., they are labeled and amplified with a universal primer or amplified with multiple primer pairs (e.g., in multiplexed form).

Analysis of cell-free circulating nucleic acids faces a number of technical challenges. For example, amplification of circulating nucleic acids in blood can be inhibited by certain components in whole blood (e.g., hemoglobin and associated iron). The devices, systems, kits, and methods disclosed herein are directed to overcoming many of these technical challenges. Furthermore, the devices, systems, kits and methods provide the following advantages: (1) minimally invasive, (2) suitable for use in homes with little or no technical training (e.g., no complex equipment required); and (3) provide information at an early stage of the condition (e.g., pregnancy, infection). Furthermore, avoiding repeated doctor/hospital visits for the purpose of blood draw and focused testing may improve patient compliance and allow for more frequent monitoring, ultimately leading to improved health outcomes at lower healthcare system costs.

In some aspects, disclosed herein are devices comprising a sample purifier for removing cells from a biological fluid sample to produce a cell-removed sample; and at least one of a detection reagent and a signal detector to detect a plurality of biomarkers in the cell removal sample. In some cases, the plurality of biomarkers comprises a plurality of cell-free DNA fragments. In some cases, each of the plurality of cell-free fragments comprises a region represented by a first sequence or a second sequence that is at least 90% homologous to the first sequence. In some cases, the first sequence is physically far enough from the second sequence that the first sequence is present on a first cell-free nucleic acid of the subject and the second sequence is present on a second cell-free nucleic acid of the subject. In some cases, the device comprises at least one nucleic acid amplification reagent and a single pair of primers capable of amplifying the first sequence and the second sequence. In some cases, at least one of the first sequence and the second sequence is repeated at least twice in the genome of the subject. In some cases, at least one of the first sequence and the second sequence is repeated at least three times in the genome of the subject. In some cases, at least one of the first sequence and the second sequence is repeated at least four times in the genome of the subject. In some cases, at least one of the first sequence and the second sequence is repeated at least five times in the genome of the subject. In some cases, the first sequence and the second sequence are each at least 10 nucleotides in length. In some cases, the first sequence is on a first chromosome and the second sequence is on a second chromosome. In some cases, the first sequence and the second sequence are on the same chromosome, but separated by at least 1 nucleotide. In some cases, the first sequence and the second sequence are functionally linked. In some cases, the first sequence is at least 80% identical to the second sequence. In some cases, the sample purifier includes a filter. In some cases, the sample purifier includes a wicking material or capillary device for pushing the biological fluid through the filter. In some cases, the filter has a pore size of about 0.05 microns to about 2 microns. In some cases, the sample purifier comprises a binding moiety that binds to a nucleic acid, a protein, a cell surface marker, or a microbubble surface marker in the fluid sample. In some cases, the binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof. In some cases, the binding moiety is capable of binding to an extracellular vesicle, wherein the extracellular vesicle is released from a fetal cell or a placental cell of the female subject. In some cases, the at least one nucleic acid amplification reagent comprises at least one isothermal amplification reagent. In some cases, the at least one isothermal amplification reagent comprises a recombinase polymerase, a single-stranded DNA binding protein, a strand-displacing polymerase, or a combination thereof. In some cases, the signal detector comprises a solid support. In some cases, the solid support is a column. In some cases, the solid support comprises a binding moiety that binds to the amplification product. In some cases, the binding moiety is an oligonucleotide. In some cases, the signal detector is a lateral flow strip. In some cases, the device comprises a detection reagent, wherein the detection reagent comprises a gold particle or a fluorescent particle. In some cases, the sample purifier removes cells from blood, and the cell-removed sample is plasma. In some cases, the device is contained in a single housing. In some cases, the apparatus operates at room temperature. In some cases, the device is capable of detecting a plurality of biomarkers in the cell removal sample within about five minutes to about twenty minutes of receiving the biological fluid. In some cases, the device comprises a transport or storage compartment. In some cases, the transport or storage compartment comprises an absorbent pad or a fluid container. In some cases, the device includes a communication connection. In some cases, the communication connection is a wireless communication system, a cable, or a cable port. In some cases, the device comprises a transcutaneous puncture device.

The method further disclosed herein includes obtaining a fluid sample from a subject, wherein the volume of the biological sample is no greater than about 300 μ L, contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sequence of interest, and detecting the presence or absence of an amplification product, wherein the presence or absence indicates a healthy state of the subject, in some cases, the fluid sample is a blood sample, in some cases, the volume of the blood sample is no greater than 120 μ l, in some cases, the fluid sample is a plasma sample from blood, in some cases, the volume of the plasma sample is no greater than 50 μ l, in some cases, the volume of the plasma sample is from about 5 μ l to about 40 μ l, in some cases, the volume of the plasma sample is from about 10 μ l to about 40 μ l, the obtaining includes performing a finger prick, in some cases, the method includes obtaining a finger prick to increase the length of the blood, the sample, the presence or the absence of a single nucleotide from a needle, the sample is a percutaneous, the presence of a blood sample, the condition is a condition of a blood sample, the condition of a blood sample is a condition of a blood sample, the condition includes the presence of a condition of a percutaneous, the condition of a blood sample is not a blood sample, the condition of a blood sample, the condition is not a blood sample is not a condition, the condition of a condition is a blood sample is not a blood sample, the condition of a blood sample is not a condition of a blood sample is not a condition, the condition, a blood sample is not a blood sample, the condition of a blood sample is not a blood sample is a blood sample is a blood sample is a blood sample is a.

By way of non-limiting example, the devices, systems, kits, and methods disclosed herein may be used to determine the gender of a fetus. The devices, systems, kits, and methods disclosed herein allow gender determination to be performed privately to the home without the need for laboratory equipment and without the risk of sample exchanges. These devices, systems, kits, and methods generally analyze cell-free fetal DNA and/or cell-free fetal RNA. The devices, systems, kits, and methods disclosed herein can advantageously determine the gender of the fetus early in pregnancy because they require little fetal nucleic acid material. The devices, systems, kits, and methods disclosed herein can provide sex status from a very small volume of sample because the devices, systems, kits, and methods are capable of detecting fragments of the Y chromosome, which comprise genes or any amplifiable region that can uniquely identify the presence or absence of the Y chromosome in a biological sample, typically in multiple copies on the Y chromosome. In most cases, the effective concentration of these fragments is higher than those gene fragments that are not present in multiple copies.

In some aspects, disclosed herein is an apparatus comprising: a sample purifier that removes cells from a fluid sample of a female subject; at least one nucleic acid amplification reagent; at least one oligonucleotide comprising a sequence corresponding to the Y chromosome, wherein the at least one oligonucleotide and nucleic acid amplification reagents are capable of producing an amplification product; and at least one of a detection reagent or a signal detector for detecting the amplification product. In some cases, the fluid sample is blood. In some cases, the sample purifier includes a filter. In some cases, the sample purifier includes a wicking material or capillary device for pushing the biological fluid through the filter. In some cases, the filter has a pore size of about 0.05 microns to about 2 microns. In some cases, the sample purifier comprises a binding moiety that binds to a nucleic acid, a protein, a cell surface marker, or a microbubble surface marker in the biological sample. In some cases, the binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof. In some cases, the binding moiety is capable of binding to an extracellular vesicle, wherein the extracellular vesicle is released from a fetal cell or a placental cell of the female subject. In some cases, the binding moiety binds to human chorionic gonadotropin protein or a transcript of a human chorionic gonadotropin-encoding gene. In some cases, the at least one oligonucleotide comprises a primer that hybridizes to a Y chromosome sequence. In some cases, the at least one oligonucleotide comprises a probe that hybridizes to a Y chromosome sequence, and wherein the probe comprises an oligonucleotide tag. In some cases, the oligonucleotide tag is not specific for a Y chromosome sequence. In some cases, the device comprises at least one primer that hybridizes to the oligonucleotide tag and produces an amplification product in the presence of the amplification reagents. In some cases, the at least one nucleic acid amplification reagent comprises at least one isothermal amplification reagent. In some cases, the at least one isothermal amplification reagent comprises a recombinase polymerase, a single-stranded DNA binding protein, a strand-displacing polymerase, or a combination thereof. In some cases, the signal detector comprises a solid support. In some cases, the solid support is a bead. In some cases, the solid support comprises a binding moiety that binds to the amplification product. In some cases, the binding moiety is an oligonucleotide. In some cases, the signal detector is a lateral flow strip. In some cases, the detection reagent comprises a gold particle. In some cases, the detection reagent comprises a fluorescent particle. In some cases, the device is contained in a single housing. In some cases, the apparatus operates at room temperature. In some cases, the device detects the amplification product within about five minutes to about twenty minutes of receiving the biological fluid. In some cases, the apparatus comprises a transport or storage compartment. In some cases, the transport or storage compartment comprises an absorbent pad or a fluid container. In some cases, the apparatus includes a communication connection. In some cases, the communication connection is a wireless communication system, a cable, or a cable port. In some cases, the apparatus includes a percutaneous puncture device.

In some aspects, disclosed herein are kits comprising the apparatus disclosed herein and a percutaneous puncture device. In some cases, the transcutaneous puncturing device is a lancet. In some cases, the device includes a capillary tube for drawing blood from a percutaneous puncture. In some cases, the kit comprises a container, pouch, wire, or cable for heating or cooling the components thereof of the device.

In some aspects, disclosed herein are methods comprising obtaining a fluid sample from a female pregnant subject, wherein the volume of the biological sample is no greater than about 300 μ L, contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sex chromosome, and detecting the presence or absence of an amplification product, wherein the presence or absence is indicative of the sex of a fetus of the female pregnant subject, in some cases, the fluid sample is a blood sample, in some cases, obtaining includes performing a finger prick, in some cases, obtaining a finger that includes squeezing a prick to increase blood from a finger prick, in some cases, obtaining the blood sample does not include performing a phlebotomy, in some cases, the fluid sample is a urine sample, in some cases, the fluid sample is a saliva sample, in some cases, the method includes removing at least one of cells, cell fragments, and microparticles from the fluid sample, in some cases, the sample includes contacting the oligonucleotide with a polymerase under conditions where the amplification reagent does not include at least one copy of the chromosome DNA, including at least one copy of the chromosome DNA, in some cases, including at least one copy of the chromosome, including at least one copy of the PCR product, in some cases, including at least one copy of the PCR amplification product, including at least one copy of a PCR amplification, under conditions where the PCR, including at least one copy of a PCR, including at least one PCR, under conditions, including at least one PCR, under conditions, under which includes a cycle, the PCR, including no amplification step, including no PCR, including at least one PCR, including no amplification step, including at least one PCR, under conditions, including at least one PCR, under which includes contacting the PCR, under which includes a PCR, under which includes contacting the PCR, under which includes a PCR, under conditions, under which includes contacting the PCR, under which includes a PCR, under conditions, under which includes a PCR, the PCR, under which includes a PCR, under which includes at least one PCR, under which includes a PCR, under which includes at least one PCR, under which includes a PCR, under conditions, the PCR, under which includes a PCR.

In some aspects, disclosed herein are methods comprising obtaining a fluid sample from a female pregnant subject with a handheld device, wherein a volume of the fluid sample is no greater than about 300 μ L, sequencing at least one cell-free nucleic acid in the fluid sample with the handheld device, detecting, by a display in the handheld device, the presence or absence of a sequence corresponding to the Y chromosome, thereby determining a gender of a fetus within the female pregnant subject, and transferring the gender to another subject with the handheld device.

In some aspects, disclosed herein are devices comprising a sample purifier for removing cells from a biological fluid sample to produce a cell-removed sample; and at least one of a detection reagent and a signal detector to detect the plurality of cell-free DNA fragments in the cell-depleted sample. In some cases, a first sequence is present on a first cell-free DNA segment of the plurality of cell-free DNA segments and a second sequence is present on a second cell-free DNA segment of the plurality of cell-free DNA segments, and wherein the first sequence is at least 80% identical to the second sequence. In some cases, the device comprises at least one nucleic acid amplification reagent and a single pair of primers capable of amplifying the first sequence and the second sequence. In some cases, at least one of the first sequence and the second sequence is repeated at least twice in the genome of the subject. In some cases, the first sequence and the second sequence are each at least 10 nucleotides in length. In some cases, the first sequence is on a first chromosome and the second sequence is on a second chromosome. In some cases, the first sequence and the second sequence are on the same chromosome, but separated by at least 1 nucleotide. In some cases, the first sequence and the second sequence are functionally linked. In some cases, the sample purifier comprises a filter, and wherein the filter has a pore size of about 0.05 microns to about 2 microns. In some cases, the filter is a vertical filter. In some cases, the sample purifier comprises a binding moiety selected from the group consisting of an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, and combinations thereof. In some cases, the binding moiety is capable of binding to an extracellular vesicle. In some cases, the at least one nucleic acid amplification reagent comprises an isothermal amplification reagent. In some cases, the signal detector is a lateral flow strip. In some cases, the device is contained in a single housing. In some cases, the apparatus operates at room temperature. In some cases, the device is capable of detecting a plurality of biomarkers in the cell removal sample within about five minutes to about twenty minutes of receiving the biological fluid. In some cases, the apparatus includes a communication connection. In some cases, the apparatus includes a percutaneous puncture device.

Further disclosed herein, in some aspects, are methods comprising obtaining a fluid sample from a subject, wherein the volume of the biological sample is no greater than about 120 microliters; contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sequence of interest to produce an amplification product; and detecting the presence or absence of the amplification product, wherein the presence or absence is indicative of a health state of the subject. In some cases, the fluid sample is a blood sample. In some cases, the fluid sample is a plasma sample from blood. In some cases, the volume of the plasma sample is no greater than 50 μ Ι. In some cases, the volume of the plasma sample is from about 10 μ Ι to about 40 μ Ι. In some cases, the sample contains about 25pg to about 250pg of total circulating cell-free DNA. In some cases, the sample contains from about 5 to about 100 copies of the sequence of interest. In some cases, the copies are at least 90% identical to each other. In some cases, the sequence of interest is at least 10 nucleotides in length. In some cases, contacting comprises performing isothermal amplification. In some cases, the contacting occurs at room temperature. In some cases, the method comprises incorporating a tag into the amplification product while the amplification occurs, and wherein detecting the presence of the amplification product comprises detecting the tag. In some cases, the tag does not comprise a nucleotide. In some cases, detecting the amplification product comprises contacting the amplification product with a binding moiety capable of interacting with the tag. In some cases, a method comprises contacting the amplification product with the binding moiety on a lateral flow device. In some cases, steps (a) through (c) are performed in less than fifteen minutes. In some cases, the method is performed by the subject. In some cases, the method is performed by an individual who has not received technical training for performing the method. In some cases, the obtaining, contacting, and detecting are performed using a single handheld device. In some cases, the health state is selected from the presence or absence of pregnancy. In some cases, the health state is selected from the presence or absence of a neurological disorder, a metabolic disorder, cancer, an autoimmune disorder, allergy, and infection. In some cases, the health state is a response to a drug or therapy.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

The novel features of the methods, devices, systems and kits disclosed herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the methods, devices, systems, and kits disclosed herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the methods, devices, systems, and kits disclosed herein are utilized, and the accompanying drawings of which:

fig. 1 shows the successful amplification and detection of single and multiple copies of a target sequence in the form of (non-fragmented) genomic DNA and cell-free DNA.

Fig. 2 illustrates an exemplary workflow of a method using the devices, systems, and kits disclosed herein.

FIG. 3 shows DNA amplification from the Y chromosome using 80 μ l of whole blood applied to the device disclosed herein.

FIG. 4 shows Y chromosomal DNA amplified by recombinase polymerase on polyacrylamide gel, specific primer sequences and amplicon sequences are listed in Table 3 and Table 4. the expected sizes, amplicons and primers are as follows, lane 1: L MW ladder, lane 2: NTC TwistDx, lane 3: PosCon TwistDx (143bp), lane 4: SRY 6-NTC, primers not shown, lane 5: SRY 6-male (370bp), primers not shown, lane 6: DYS 14-Amp 10(134bp), primers DYS14_1_ F _ L ong and DYS14_5_ R _ L g, lane 7: DYS 7-Amp 11(148bp), primers DYS14_5_ F _ L ong and DYS L _ R _ L g, lane 8: TTTY L-Amp 10(118bp), primers DYS14_5_ F _ L-AMT _ L bp), primers TTTY L-ATTY-L primer TTTY-SRY-L-ATTY-L bp, primers TTTY-L-ATP-ATTty-L-L bp, primers TTTY-L-ATTty-L-ATP-ATTty-ATP-L-ATTty-L-Tty-L-Tty-L-Tty-L-primer-Tty-primer-Tty-L-Tty-L-primer-Tty-.

Figure 5 shows real-time detection of DYS 14Y-chromosome amplification product from recombinase polymerase amplification.

Figure 6 shows real-time detection of DYS 14Y chromosome amplification product from recombinase polymerase amplification versus a female control sample.

FIG. 7 shows lateral flow detection of the product of the Y-chromosome DYS14 recombinase polymerase amplification.

Fig. 8 illustrates an example of how a mobile device may be used to display, interpret and/or share results obtained from the devices and methods disclosed herein. Fig. 8A shows an overview of the functionality of a mobile application that may be used in connection with the devices, systems, and kits disclosed herein. FIG. 8B illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a stepwise flow (walkthrough) to guide the user through the use of the devices, systems, and kits disclosed herein. FIG. 8C illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a home screen allowing the user to access the mobile application functionality disclosed herein. FIG. 8D illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a progress chart informing the user of the status of the process to connect to the device, system or kit disclosed herein to receive information. FIG. 8E illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a gender testing report to the user. FIG. 8F illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a social sharing screen that allows the user to access features to share gender test results. FIG. 8G illustrates a non-limiting example of a graphical user interface for a mobile application; in this case, the interface provides a home screen allowing the user to access other features such as a pregnancy log and a timeline of important pregnancy related events.

Fig. 9 shows an agarose gel of RPA products generated against the TSPY1(DYS14) locus on the Y chromosome.

FIG. 10 shows a nucleic acid lateral flow immunoassay strip with human TSPY1(DYS14) Y chromosome RPA-L F product.

Figure 11 shows generation of amplicons from highly repetitive Y chromosome regions (HRYR) with samples from male humans.

Figure 12 shows that amplicons from highly repetitive Y chromosome regions (HRYR) were not generated with samples from female humans.

FIG. 13 illustrates the use of Vivid^TMComparison of membranes with plasma isolated by standard centrifugation methods from less than 50 microliters (μ l) of male whole blood.

Figure 14 shows the yields of bead-based and column-based purification using 20 μ Ι human plasma as input for extraction from male and female subjects.

Fig. 15A-15C illustrate an exemplary device disclosed herein. Fig. 15A shows a side view of an exemplary device. Fig. 15B shows a top view of an exemplary device. Fig. 15C illustrates a front view of an exemplary device.

Certain terms

The following description is provided to aid in understanding the methods, systems, and kits disclosed herein. The following description of terms used herein is not intended to limit the definition of those terms. These terms are further described and exemplified throughout this application.

Generally, the terms "cell-free polynucleotide" and "cell-free nucleic acid" are used interchangeably herein and refer to a polynucleotide or nucleic acid that can be isolated from a sample without extracting the polynucleotide or nucleic acid from the cell. A cell-free nucleic acid is a nucleic acid that is not contained within a cell membrane, i.e., it is not encapsulated within a cell compartment. In some embodiments, cell-free nucleic acids are nucleic acids that are not bound by a cell membrane and circulate or are present in blood or other fluids. In some embodiments, the cell-free nucleic acid is cell-free prior to and/or after collection of a biological sample containing it, and is not released from the cells as a result of manipulation of the sample by human, deliberate, or other means, including manipulation at or after collection of the sample. In some cases, cell-free nucleic acids are produced in a cell and released from the cell by physiological means including, for example, apoptotic and non-apoptotic cell death, necrosis, autophagy, spontaneous release (e.g., of DNA/RNA-lipoprotein complexes), secretion, and/or mitotic collapse. In some embodiments, cell-free nucleic acids include nucleic acids released from cells by biological mechanisms (e.g., apoptosis, cell secretion, vesicle release). In further or additional embodiments, the cell-free nucleic acid is not a nucleic acid extracted from a cell by human manipulation of the cell or sample processing (e.g., cell membrane disruption, lysis, vortexing, shearing, etc.).

In some cases, the cell-free nucleic acid is cell-free fetal nucleic acid. Generally, the term "cell-free fetal nucleic acid" as used herein refers to cell-free nucleic acid as described herein, wherein the cell-free nucleic acid is from a cell comprising fetal DNA. Typically, a significant fraction of cell-free fetal nucleic acid is found in maternal biological samples due to the regular shedding of placental tissue during pregnancy in pregnant subjects. Typically, many of the cells in the placental tissue casts are cells that contain fetal DNA. Thus, in some cases, cell-free fetal nucleic acid is nucleic acid released from placental cells.

In some cases, cellular nucleic acids (nucleic acids contained by a cell) are intentionally or unintentionally released from a cell by the devices and methods disclosed herein. However, these are not considered "cell-free nucleic acids," as that term is used herein. In some cases, the devices, systems, kits, and methods disclosed herein provide for analyzing cell-free nucleic acids in a biological sample, and in the process, also cellular nucleic acids.

As used herein, the term "cellular nucleic acid" refers to a polynucleotide contained in a cell. Cellular nucleic acids can be described as nucleic acids that can be released from cells as a result of manipulation of a biological sample. Non-limiting examples of manipulation of a biological sample include centrifugation, vortexing, shearing, mixing, lysing, and adding to the biological sample reagents (e.g., detergents, buffers, salts, enzymes) that are not present in the biological sample at the time of acquisition. Cellular nucleic acids can be described as nucleic acids that can be released from cells as a result of lysis conditions (e.g., shear, lysis buffer). Cellular nucleic acids can be described as nucleic acids that can be released from cells as a result of contacting a biological sample with a lysis reagent. Exemplary lysis reagents are disclosed herein. In some cases, cellular nucleic acids are nucleic acids released from cells as a result of mechanical, human, or robotic disruption or lysis of the cells.

As used herein, the term "biomarker" generally refers to any marker of a biological or condition of a subject. A biomarker may be an indicator or result of a disease or condition. The biomarker may be an indicator of health. The biomarker may be an indicator of a genetic abnormality or a genetic condition. The biomarker may be a circulating biomarker (e.g., found in a biological fluid such as blood). The biomarker may be a tissue biomarker (e.g., found in a solid organ such as liver or bone marrow). Non-limiting examples of biomarkers include nucleic acids, epigenetic modifications, proteins, peptides, antibodies, antibody fragments, lipids, fatty acids, sterols, polysaccharides, carbohydrates, viral particles, microbial particles. In some cases, the biomarker may even include whole cells or cell fragments.

As used herein, the term "genetic information" generally refers to one or more nucleic acid sequences. In some cases, the genetic information may be a single nucleotide or amino acid. For example, the genetic information may be the presence (or absence) of a single nucleotide polymorphism. The term "genetic information" may also refer to epigenetic modification patterns, gene expression data, and protein expression data, unless otherwise specified. In some cases, the presence, absence, or amount of a biomarker provides genetic information. For example, cholesterol levels may indicate the genetic form of hypercholesterolemia. Thus, the genetic information should not be limited to nucleic acid sequences.

As used herein, the term "genomic equivalent" generally refers to the amount of DNA that must be present in a purified sample in order to ensure that all genes will be present.

As used herein, the terms "clinical," "clinical environment," "laboratory" or "laboratory environment" refer to a hospital, clinic, pharmacy, research facility, pathology laboratory, or other commercial environment where trained personnel are employed to process and/or analyze biological and/or environmental samples. These terms are in contrast to point of care (point of care), remote locations, homes, schools, and other non-commercial, non-institutional environments.

As used herein, the term "about" with respect to a number indicates a range that includes the number plus or minus 10% of the number. The term "about" with respect to a range of values means that the range minus 10% of its lowest value and plus 10% of its highest value.

As used herein, the term "specific" refers to a sequence or biomarker that is found only in, on, or at the point where the sequence or biomarker is specific. For example, if the sequence is specific for the Y chromosome, this means that it is found only on the Y chromosome, but not on the other chromosome.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, including mixtures thereof.

As used herein, the terms "homology", "homology" or "percent homology" describe the sequence similarity of a first amino acid sequence or nucleic acid sequence with respect to a second amino acid sequence or nucleic acid sequence, in some cases, the homology may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268,1990, as in the case of a sequence alignment of at least 20% or more, as in the case of a sequence alignment of at least 10% or more, as in the case of a sequence alignment of at least 10% of a sequence homology, as in a sequence alignment of at least 10% of a sequence alignment of at least 10% or 70, as in the case of a sequence alignment of at least 10% or more, as in a sequence alignment of at least 10, as in a sequence alignment of at least 10, or 70, or 10, as in a sequence homology window of at least 10, as in a sequence alignment of at least 10, or a sequence alignment of at least 10, or 70, as in a sequence alignment of a sequence homology window of at least 10, or a sequence homology or a sequence alignment of at least 10, or a sequence homology window of at least 10, or a sequence alignment of at least 10, or a sequence homology window of at least 10, or a sequence homology or a sequence alignment of at least 10, as in a sequence alignment of at least 10, or a sequence alignment of a sequence homology window of a sequence alignment of a sequence homology or a sequence alignment of at least 10, or a sequence homology window of at least 10, or a sequence homology window of a sequence alignment of at least 10, or a sequence homology window, or a sequence homology or a sequence, as in a sequence homology or a sequence homology window of a sequence homology window, or a sequence alignment of a sequence homology or a sequence homology window of a sequence homology or a sequence alignment of at least 10, or a sequence alignment of a sequence homology window of at least 10, or a sequence homology or a sequence, or a sequence alignment of a sequence, or a sequence homology window of at least 10, or a sequence alignment of a sequence homology window of at least 10, or a sequence homology window of a sequence alignment of a sequence homology of at least 10, or a sequence homology window, or a sequence homology or a sequence, or a sequence alignment of at least 10, or a sequence homology or a sequence alignment of at least 10, or a sequence, as in a sequence, or a sequence alignment of a sequence, or a sequence alignment of at least 10, or a sequence homology window, or a sequence alignment of at least 10, or a sequence alignment of at least 10, or a sequence alignment of at least 10, as in a sequence alignment of at least 10, or a sequence alignment of at least 10, or a sequence homology window of a sequence alignment of at least 10, or a sequence alignment of at least 10, or a sequence homology or a sequence, or a sequence.

Throughout this application, the phrases "nucleic acid corresponding to chromosome" and "sequence corresponding to chromosome", for example, "nucleic acid corresponding to Y chromosome" and "sequence corresponding to Y chromosome", are recited. As used herein, these phrases are intended to convey that "nucleic acid corresponding to a chromosome" is represented by a nucleic acid sequence that is identical or homologous to a sequence found in that chromosome. The term "homologous" is described in the foregoing description.

Throughout this application, chromosomal locations are described. These position numbers refer to the genomic version (GenomeBuild) hg38(UCSC) and GRCh38 (NCBI). The genomic version may also be referred to in the art as a reference genome or reference component. It may be derived from a plurality of subjects. It should be understood that there are multiple reference components available and that more may be generated over time. However, one skilled in the art will be able to determine the relative positions provided herein in another genomic version or a reference genome.

Detailed Description

Genetic testing is traditionally performed in a laboratory or clinical setting. However, in many cases where genetic testing may be useful, access to a laboratory or clinic is not possible or practical. Therefore, it is desirable to have genetic testing operable at a point of need (e.g., a location remote from laboratories and clinics). Genetic testing for performance at a point of need (e.g., home, school, farm) is preferably cost effective and simple to operate by untrained personnel. Genetic testing at the point of need preferably requires only a small amount of biological sample. Traditionally, genetic testing requires venous blood draw (phlebotomy) to obtain milliliters of blood containing sufficient DNA for analysis. However, phlebotomy is impractical at the point of need. Ideally, genetic testing requires only the amount of blood obtained by drawing capillary blood, such as by finger prick. This means that there is a need to design point-of-demand devices and methods for genetic testing to function when the sample input is low and the abundance of target molecules intended for detection is low.

To illustrate the scale challenge of analyzing circulating nucleic acids in capillary blood using a home or point of demand device, cell-free DNA analysis may be used for the purpose of determining fetal gender. Traditionally, this is done by drawing venous blood of eight milliliters of blood. Up to four milliliters of plasma may be obtained from eight milliliters of blood draw. The amount of capillary blood from the finger prick (about 20 μ l) was about 1/400 of the blood draw. On average, four thousand genome equivalents (or genome copies) appear to be present in the form of circulating cell-free DNA in four milliliters of plasma. Accordingly, the amount of finger stick will contain only about ten genome equivalents (or copies). In pregnant women, on average 10% of the circulating cell-free DNA is derived from the fetus. Thus, a venous blood sample will have an average of four hundred fetal genomic copies, and a finger prick sample will have only an average of 1 fetal genomic copy. It is evident from these calculations that the assay performance (e.g., sensitivity) of any assay directed to a single genomic region will be limited by statistical sampling. For example, attempting to detect fetal gender in a pregnant female from a finger prick volume of blood using a genomic region that occurs only once and is a single target region (e.g., using the SRY gene) would require at least 120 μ Ι of capillary blood to exhibit at least 1 copy of the target region in 95% of all test samples.

In addition to low input of adaptation samples, it is also desirable to have genetic tests capable of analyzing circulating cell-free nucleic acids (DNA and RNA), e.g., circulating cell-free fetal DNA, circulating tumor DNA, circulating DNA from transplanted donor organs, and circulating DNA released from specific tissues as part of health-related issues, disease progression, or therapeutic response. However, analysis of circulating cell-free nucleic acids is challenging due to their shorter half-life and consequent lower abundance. Furthermore, if the sample is not taken care of to avoid leukocyte lysis, the DNA released by the leukocytes may dilute the circulating cell-free nucleic acids in the blood. Leukocyte DNA produces background noise during detection of circulating cell-free nucleic acids, which reduces the sensitivity and specificity of the assay.

The devices, systems, kits, and methods disclosed herein overcome these challenges by combining gentle and efficient processing of small sample volumes (e.g., less than 1ml) with unique target region selection and assay designs that take advantage of the highly fragmented nature of circulating cell-free dna (cfdna). For example, the devices, systems, kits, and methods disclosed herein can provide reliable genetic information from a single finger prick. The devices, systems, kits, and methods disclosed herein provide for analysis of multiple target regions along a target gene that are spaced far enough apart that when the target gene is fragmented in the circulation, the target regions may be physically separated. Thus, while the above-described limitations of statistical sampling exist for single long DNA fragments traditionally analyzed in gene testing, variations in sampling statistics are advantageous for cfDNA fragments. Although only 1 total genome equivalent may be present in a capillary blood sample, there are many individual cfDNA fragments. Thus, if multiple target regions on individual cell-free fragments are analyzed, sensitive amplification can be achieved from low input.

FIG. 1 shows the success rate of detecting multiple target regions when detecting or amplifying genomic DNA versus circulating cell-free DNA. If only a single copy region or target sequence is amplified, a relatively high amount of blood is required to ensure that at least 95% of the samples have 1 copy (which is the minimum required for any amplification or detection opportunity). If multiple copies of a region or copies (e.g., 20 copies) of a target sequence are amplified, but they are present in the sample as long DNA molecules, such as genomic DNA, a relatively high amount of blood is still required to detect at least 1 copy of the target sequence. In contrast, the success rate of amplification and detection of target regions in cell-free DNA from relatively low amounts of sample is significantly improved. By way of non-limiting example, a relatively low amount would be the amount of blood from a finger prick, while a relatively high amount would be the amount of blood from a phlebotomy.

As an example, if there are twenty target regions along a genomic region and they are spaced far enough apart that they can be analyzed and detected independently when DNA is fragmented, the input volume required to have at least 1 target region in 95% of all samples goes from 140 μ Ι (genomic DNA) to less than 25 μ Ι (cfdna), significantly improving sensitivity. In some cases, the target region contains the same sequence or similar sequences. These target regions may be referred to as copies. A non-limiting example of this is the TTTSY region on the Y chromosome, which has about 20 homologs. This is an example of a highly repetitive region, described further herein. Advantageously, all twenty regions can be amplified with the same primer pair. The concentration of fragments containing the target region is 20 times higher than that of fragments of the non-fragmented Y chromosome or non-duplicated Y chromosome. Thus, the signal from the TTTSY region will be twenty times more than that obtained from the unfragmented Y chromosome or the non-repetitive region of the Y chromosome. Another way to look at is that the likelihood of one copy of the target region being present in a low volume sample is twenty times greater than a non-target region that is not repeated or does not share some detectable commonality with another region.

In other cases, the target regions may not share similar sequences, but share another characteristic such as a similar epigenetic state. For example, multiple target regions may have different sequences, yet they are hypermethylated. Regardless of the specific type of epigenetic modification, the sites of epigenetic modification are spaced appropriately to exploit the fragmentation pattern of circulating cell-free DNA that produces a number of circulating cfDNA fragments, at least one of which can be detected in a small volume. By way of non-limiting example, bisulfite sequencing can be used to detect selected target regions that are far enough from each other to be located on separate cfDNA fragments and are all hypermethylated when the subject has cancer. In small sample volumes (e.g., finger prick blood), the likelihood of all these fragments (equivalent to non-fragmented DNA) being present is low, whereas the likelihood of at least one fragment being present is high, and cancer can be detected.

In other cases, the target region may not contain similar sequences, and may not contain similar epigenetic states. In this case, detection may require multiple primer sets or library preparation followed by amplification with universal primers to detect several different target regions. By way of non-limiting example, detection of fetal RHD genes in RHD-negative pregnant women from finger-pricked volume of blood can be achieved by detecting multiple different exons of the RHD gene in cell-free fetal DNA fragments using multiple sets of primers. By using twenty sets of primers, the same sensitivity as that achieved using twenty repeat sequences in the example of TTTSY region described above can be achieved. Sensitivity can be increased by selecting primers that amplify regions that are physically distant in the RHD gene and therefore likely to be present on different cell-free DNA fragments. Detection of the fetal RHD gene in RHD-negative pregnant women is important to prevent hemolytic disease of the newborn caused by the mother's antibody to the child's blood. Currently, RHD testing is today performed by whole blood withdrawal (eight milliliters of blood) to achieve reasonably reliable results. This volume is considered necessary to achieve reliable results because it is based on the possibility that the entire RHD gene will be present in the sample. Based on this assumption, the probability of obtaining the complete RHD gene in the amount of blood that is pricked by a finger is low and will easily lead to false negative results.

Regardless of how the target regions are selected, these regions are present in the sample as individual biomarkers when amplification or detection is performed on cell-free fragmented DNA. The concentration of fragments containing the target region is greater than the concentration of corresponding non-fragmented DNA or fragments that cannot be measured as a group. Thus, there will be more signal from the target region than would be obtained from either un-fragmented DNA or from one copy of the assay target region. The presence of a target region is more likely to be detected in a low volume sample than a non-target region that is not repeated or does not share some commonality with another region. By assaying multiple target regions in multiple DNA fragments, assay sensitivity is improved over traditional assays.

Blood is a reliable source of cell-free nucleic acids. Most methods for analyzing cell-free nucleic acids from blood involve the separation of plasma or serum fractions containing cell-free nucleic acids. The devices, systems, kits, and methods disclosed herein allow for gentle processing of blood samples at the point of need. This may avoid, prevent or reduce leukocyte lysis. The devices, systems, kits, and methods disclosed herein allow for rapid processing of blood samples at points of demand. This avoids prolonged storage and shipment of samples that may lead to lysis of blood cells. In some cases, the devices disclosed herein perform integrated separation, e.g., immediate separation of plasma by filtration, to avoid, reduce, or prevent cell lysis. When reagents (e.g., probes, primers, antibodies) or detection methods do not provide much specificity, it may be desirable to immediately separate cells from cfDNA. In some cases, the method is performed with whole blood to avoid any leukocyte lysis. When a relatively high specificity can be achieved, it may be more desirable to perform the assay from whole blood.

In addition to requiring only a small volume of sample, the devices, systems, kits, and methods disclosed herein are highly desirable for at least the following reasons. The devices, systems, kits, and methods disclosed herein typically require little or no technical training. Thus, the cost of performing genetic testing is reduced relative to the cost of testing performed by trained personnel, and the test can be used for subjects who are not accessible to trained personnel. Furthermore, results can be obtained in minutes (e.g., less than an hour). This may be particularly important when testing for infection. Individuals or animals that test positive for infection can be quickly isolated and treated to prevent spread of infection. Furthermore, the results can be obtained privately. In some cases, only the patient undergoing the test is informed of the acquired genetic information. The devices, systems, and kits disclosed herein are generally lightweight and handheld, making them suitable and useful in remote locations. Thus, they may be used at home, school, battlefield, farm, or any other location where it is impractical or inconvenient to access a laboratory or clinical setting. Furthermore, because the samples can be analyzed at the point of care, there is no need to store or ship the samples, reducing the risk of sample degradation and misidentification (e.g., sample exchange).

In some cases, the devices, systems, kits, and methods disclosed herein are desirable because the genetic information may be kept secret from the user. In fact, even the use of the device can be kept secret. Alternatively, the devices, systems, kits, and methods are configured to share information with others, or can be easily adjusted by a user to share information (e.g., turn on a bluetooth signal). For example, information can be easily shared with a nurse or doctor. In some cases, the device or system may send/share test results to healthcare practitioners or staff at the office or hospital through a security portal or Application Programming Interface (API). In some cases, the user may choose to share information with the healthcare practitioner in person after receiving the results. In some cases, information may even be shared in real-time. For example, gender determination can be shared with family and friends in real time via the communication components of the devices, systems, and kits. Such communication is desirable for couples or families that are separated by, for example, military missions, employment obligations, immigration policies, or health concerns. In the example of gender determination provided above, a pregnant female may be able to conduct a video conference (e.g., Skype, Face Time) with her husband overseas, under the privacy of her own home, to simultaneously determine the gender of her child.

The devices, systems, kits, and methods disclosed herein have many applications. The devices, systems, kits, and methods disclosed herein allow for diagnosis and monitoring of medical conditions. Non-limiting examples of medical conditions include autoimmune conditions, metabolic conditions, cancer, and neurological conditions. The devices, systems, kits, and methods disclosed herein allow for personalized medication, including microbiome testing, determination of appropriate personal medical doses, and/or detection of responses to drugs or their doses. The devices, systems, kits and methods disclosed herein also allow for the detection of food allergens and the detection of food/water contamination. The devices, systems, kits, and methods disclosed herein provide for the detection of infection by a pathogen and/or resistance of a subject to drugs that can be used to treat the infection. In almost all cases, little or no technical training or large, expensive laboratory instrumentation is required.

I.Device, system and kit

In some aspects, disclosed herein are devices, systems, and kits for obtaining genetic information. As described herein, the devices, systems, and kits disclosed herein allow a user to collect and test a sample at a selected location to determine the presence and/or quantity of a target analyte in the sample. The sample may be a sample from a subject, such as a biological fluid (e.g., blood, urine). The sample may be an environmental sample (e.g., wastewater, soil, food/beverage).

In some cases, the devices, systems, and kits disclosed herein comprise a sample purifier that removes at least one biological component from a biological fluid sample of a subject; at least one nucleic acid amplification reagent; at least one oligonucleotide comprising a sequence corresponding to a region of interest, wherein the at least one oligonucleotide and nucleic acid amplification reagents are capable of producing an amplification product; and at least one of a detection reagent or a signal detector for detecting the amplification product. In some cases, the at least one biological component is a cell, cell fragment, microparticle, exosome, nucleosome, protein, or a combination thereof. By way of non-limiting example, the subject may be a pregnant subject and the region of interest may be a region on the Y chromosome. By way of non-limiting example, the region of interest can be a gene involved in cancer, an autoimmune condition, a neurological disorder, a metabolic disorder, a cardiovascular disease, immunity (e.g., susceptibility to infection or resistance to drug), and drug metabolism. A gene involved in a disease, disorder or condition is considered to be a gene that, when mutated, deleted, duplicated, epigenetically modified, underexpressed or overexpressed, alters at least one of the symptoms, outcome, duration or onset of the disease, disorder or condition.

In some cases, the devices, systems, and kits disclosed herein comprise a sample purifier that removes cells from a biological fluid sample of a subject; at least one nucleic acid amplification reagent; at least one oligonucleotide comprising a sequence corresponding to a region of interest, wherein the at least one oligonucleotide and nucleic acid amplification reagents are capable of producing an amplification product; and at least one of a detection reagent or a signal detector for detecting the amplification product. In some cases, the devices, systems, and kits disclosed herein comprise a miniaturized digital nucleic acid amplification platform. By way of non-limiting example, a miniaturized nucleic acid amplification platform may be located on a chip within the device disclosed herein, thereby keeping the entire device or system in a handheld size (e.g., similar to a cell phone). In some cases, a miniaturized nucleic acid amplification platform incorporates or accompanies digital output in order to display test results.

In some cases, the devices, systems, and kits disclosed herein comprise a sample purifier that removes cells from a biological sample of a subject; a nucleic acid sequencer for obtaining sequencing reads from nucleic acids in a biological sample; and at least one of a detection reagent or a signal detector for detecting sequencing reads. Non-limiting examples of nucleic acid sequencers include next generation sequencing machines, nanopore sequencers, single molecule counters (e.g., counting barcode/tag labeled sequences).

In general, the devices, systems, and kits disclosed herein incorporate a variety of functions, such as, for example, purification, amplification, detection, and assay of a target analyte (including amplification products thereof), and combinations thereof. In some cases, multiple functions are performed in a single assay assembly unit or a single device. In some cases, all functions occur outside of a single unit or device. In some cases, at least one function occurs outside of a single unit or device. In some cases, only one function occurs outside of a single unit or device. In some cases, the sample purifier, nucleic acid amplification reagents, oligonucleotides, and detection reagents or components are housed in a single device. In general, the devices, systems, and kits disclosed herein comprise a display, a connection to or communication with the display, for relaying information about a biological sample to one or more persons.

In some cases, the devices, systems, and kits comprise additional components disclosed herein. Non-limiting examples of additional components include a sample transport compartment, a sample storage compartment, a sample and/or reagent receptacle, a temperature indicator, an electronic port, a communication connection, a communication device, a sample collection device, and a housing unit. In some cases, the add-on component is integrated with the device. In some cases, the add-on component is not integrated with the device. In some cases, the add-on components are housed in a single device with the sample purifier, nucleic acid amplification reagents, oligonucleotides, and detection reagents or components. In some cases, the add-on components are not housed in a single device.

In some cases, the devices, systems, and kits comprise a receptacle for receiving a biological sample. The receptacle may be configured to hold 1 μ Ι to 1ml of the biological sample. The receptacle may be configured to hold 1 μ Ι to 500 μ Ι of biological sample. The receptacle may be configured to hold 1 μ Ι to 200 μ Ι of biological sample. The receptacle may have the same defined volume as the appropriate volume of sample for processing and analysis by the remaining device/system components. This would eliminate the need for a user of the device, system or kit to measure a specified volume of sample. The user will only need to fill the receptacle, thereby ensuring that the appropriate volume of sample is delivered to the device/system. In some cases, the devices, systems, and kits do not comprise a receptacle for receiving a biological sample. In some cases, the sample purifier receives the biological sample directly. Similar to the description of the receptacle above, the sample purifier may have a defined volume suitable for processing and analysis by the remaining device/system components.

Generally, the devices, systems, and kits disclosed herein are intended to be used entirely at the point of care. However, in some cases, the user may wish to save or send the analyzed sample to another location (e.g., laboratory, clinic) for additional analysis or confirmation of results obtained at the point of care. In some cases, devices, systems, and kits include a transport compartment or a storage compartment for these purposes. The transport compartment or storage compartment may be capable of containing a biological sample, a component thereof, or a portion thereof. The transport compartment or storage compartment may be capable of containing the biological sample, a portion thereof, or a component thereof during shipment to a location remote from the immediate user. A non-limiting example of a location remote from the end user may be a laboratory or clinic when the end user is at home. In some cases, the home has no machinery or additional equipment to perform additional analyses on the biological sample. The transport compartment or storage compartment may be capable of containing the products of a reaction or process occurring in the apparatus. In some cases, the product of the reaction or process is a nucleic acid amplification product or a reverse transcription product. In some cases, the product of the reaction or process is a component of the biological sample that binds to a binding moiety described herein. Biological sample components may include nucleic acids, cell fragments, extracellular vesicles, proteins, peptides, sterols, lipids, vitamins, or glucose, any of which may be analyzed at a remote location of a user. In some cases, the transport compartment or storage compartment comprises an absorbent pad, paper, glass container, plastic container, polymer matrix, liquid solution, gel, preservative, or a combination thereof. In some cases, the device, system, or kit comprises a stabilizer (chemical substance or structure (e.g., matrix)) that reduces enzyme activity during storage and/or transport.

In general, the devices and systems disclosed herein are portable to a single person. In some cases, the devices and systems are handheld. In some cases, the devices and systems have a maximum length, a maximum width, or a maximum height. In some cases, the devices and systems are housed in a single unit having a maximum length, a maximum width, or a maximum height. In some cases, the maximum length is no greater than 12 inches. In some cases, the maximum length is no greater than 10 inches. In some cases, the maximum length is no greater than 8 inches. In some cases, the maximum length is no greater than 6 inches. In some cases, the maximum width is no greater than 12 inches. In some cases, the maximum width is no greater than 10 inches. In some cases, the maximum width is no greater than 8 inches. In some cases, the maximum width is no greater than 6 inches. In some cases, the maximum width is no greater than 4 inches. In some cases, the maximum height is no greater than 12 inches. In some cases, the maximum height is no greater than 10 inches. In some cases, the maximum height is no greater than 8 inches. In some cases, the maximum height is no greater than 6 inches. In some cases, the maximum height is no greater than 4 inches. In some cases, the maximum height is no greater than 2 inches. In some cases, the maximum height is no greater than 1 inch.

Sample collection

In some cases, the devices, systems, and kits disclosed herein comprise a sample collector. In some cases, the sample collector is provided separately from the rest of the device, system, or kit. In some cases, the sample collector is physically integrated with the device, system, or kit or components thereof. In some cases, the sample collector is integrated with a receptacle described herein. In some cases, the sample collector may be a cup, tube, capillary, or well for applying biological fluids. Biological fluids are described herein and throughout. In some cases, the sample collector may be a cup for applying urine. In some cases, the sample collector may comprise a pipette for applying urine in the cup to a device, system, or kit. In some cases, the sample collector may be a capillary tube for applying blood integrated with the devices disclosed herein. In some cases, the sample collector may be a tube, well, pad, or paper for applying saliva integrated with the devices disclosed herein. In some cases, the sample collector may be a pad or paper for applying sweat.

In some cases, the devices, systems, and kits disclosed herein comprise a percutaneous puncture device. Non-limiting examples of transcutaneous puncturing devices are needles and lancets. In some cases, the sample collector comprises a transcutaneous puncture device. In some cases, the devices, systems, and kits disclosed herein comprise microneedles, microneedle arrays, or microneedle patches. In some cases, the devices, systems, and kits disclosed herein comprise hollow microneedles. By way of non-limiting example, the transcutaneous puncture device is integrated with a hole or capillary such that when a subject pierces their finger, blood is released into the hole or capillary where it will be available to the system or apparatus for analysis of its components. In some cases, the transcutaneous puncturing device is a push button device having a needle or lancet in a concave surface. In some cases, the needle is a microneedle. In some cases, the percutaneous puncture device comprises an array of microneedles. By pressing an actuator, button or position on the non-needle side of the concave surface, the needle pierces the subject's skin in a more controlled manner than a lancet. In addition, the button device may contain a vacuum source or plunger to assist in drawing blood from the puncture site.

In some cases, the devices disclosed herein comprise a percutaneous puncture device, wherein the device stabilizes blood. The device containing the stabilized blood or portions thereof (e.g., storage/shipping chambers, filter pads, or paper) may be sent to a laboratory for additional processing and analysis. In some cases, the devices disclosed herein comprise a percutaneous puncture device, wherein the device comprises a sample purifier that separates plasma from red blood cells. The device containing the plasma or a portion thereof may be sent to a laboratory for additional processing and analysis.

Sample purification

Disclosed herein are devices, systems, and kits comprising a sample purifier to remove unwanted materials or non-target components of a biological sample to modify the sample. Depending on the source of the biological sample, the unwanted substances may include, but are not limited to, proteins (e.g., antibodies, hormones, enzymes, serum albumin, lipoproteins), free amino acids and other metabolites, microbubbles, nucleic acids, lipids, electrolytes, urea, urobilins, drugs, mucus, bacteria and other microorganisms and combinations thereof. In some cases, the sample purifier separates components of a biological sample disclosed herein. In some cases, the sample purifiers disclosed herein remove sample components that would inhibit, interfere with, or otherwise be detrimental to subsequent processing steps, such as nucleic acid amplification or detection. In some cases, the resulting modified sample is enriched for the target analyte. This can be considered an indirect enrichment of the target analyte. Alternatively or additionally, the target analyte may be captured directly, which is considered to be a direct enrichment of the target analyte.

In some cases, the sample purifier includes a separation material for removing unwanted material other than patient cells from the biological sample. Useful separation materials can include specific binding moieties that bind or associate with a substance. The binding may be covalent or non-covalent. Any suitable binding moiety known in the art for removing a particular substance may be used. For example, antibodies and fragments thereof are often used to remove proteins from a sample. In some cases, a sample purifier disclosed herein comprises a binding moiety that binds to a nucleic acid, a protein, a cell surface marker, or a microbubble surface marker in a biological sample. In some cases, the binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof.

In some cases, a sample purifier disclosed herein comprises a filter. In some cases, a sample purifier disclosed herein comprises a membrane. Typically, the filter or membrane is capable of isolating or removing cells, cell particles, cell fragments, blood components other than cell-free nucleic acids, or a combination thereof from a biological sample disclosed herein.

In some cases, the sample purifier facilitates separation of plasma from cellular components of the blood sample prior to initiating the molecular amplification reaction. Plasma separation can be achieved by several different methods, such as centrifugation, sedimentation or filtration. In some cases, a sample purifier disclosed herein comprises a filter. In some cases, the sample purifier employs vertical filtration. Vertical filtration is filtration driven by capillary forces for separating plasma from blood. In some cases, the sample purifier includes a filter matrix for receiving whole blood, the filter matrix having a pore size that prevents passage of cells, while plasma can pass through the filter matrix without inhibition. In some cases, the filtration matrix combines large pore sizes at the top of the filter with small pore sizes at the bottom of the filter, resulting in very gentle treatment of the cells, preventing cell degradation or lysis during filtration. This is advantageous because cellular degradation or lysis will result in the release of nucleic acids from blood cells or maternal cells, which will contaminate the target cell-free nucleic acid. Non-limiting examples of such filters include Pall Vivid^TMGR films, Munktell Ahlstrom filters (see, e.g., W)O2017017314), TeraPore Technologies filters.

In some cases, the sample purifier comprises a suitable separation material, such as a filter or membrane, that removes unwanted material from the biological sample without removing cell-free nucleic acids. In some cases, the separation material separates substances in the biological sample based on size, e.g., the separation material has a pore size that excludes cells but is permeable to cell-free nucleic acids. Thus, when the biological sample is blood, plasma and/or serum can move through the separation material in the sample purifier faster than the blood cells, and the plasma or serum containing any cell-free nucleic acids permeates into the pores of the separation material. In some cases, the biological sample is blood and the cells slowed and/or captured in the separation material are red blood cells, white blood cells, or platelets. In some cases, the cells are from tissue that is in contact with a biological sample in vivo, including but not limited to bladder or urothelial cells (in urine) or buccal cells (in saliva). In some cases, the cell is a bacterium or other microorganism.

In some cases, the sample purifier is capable of slowing and/or capturing cells without damaging the cells, thereby avoiding release of cellular contents including cellular nucleic acids and other proteins or cellular fragments that may interfere with subsequent evaluation of cell-free nucleic acids. This may be achieved, for example, by a gradual, progressive reduction in aperture along the path of the lateral flow strip or other suitable assay format to allow cell movement to be gently slowed, thereby minimizing forces on the cells. In some cases, at least 95%, at least 98%, at least 99%, or 100% of the cells in the biological sample remain intact when captured in the separation material. In addition to or independent of size separation, the separation material can capture or separate unwanted substances based on properties of the cell other than size, e.g., the separation material can comprise a binding moiety that binds to a cell surface marker. In some cases, the binding moiety is an antibody or antigen-binding antibody fragment. In some cases, the binding moiety is a ligand or receptor binding protein directed to a receptor on a blood cell or microvesicle.

In some cases, the devices, systems, and kits disclosed herein employ vertical filtration driven by capillary forces to separate components or fractions from a sample (e.g., separating Plasma from blood). by way of non-limiting example, vertical filtration may include gravity-assisted Plasma Separation.

The sample purifier may comprise a lateral filter (e.g., the sample does not move in the direction of gravity or the sample moves perpendicular to the direction of gravity). The sample purifier may comprise a vertical filter (e.g., the sample moves in the direction of gravity). The sample purifier may comprise a vertical filter and a lateral filter. The sample purifier may be configured to receive the sample or portion thereof through a vertical filter followed by a lateral filter. The sample purifier may be configured to receive the sample or portion thereof through a lateral filter followed by a vertical filter. In some cases, the vertical filter comprises a filtration matrix. In some cases, the filtration matrix of the vertical filter comprises pores having a pore size that prevents the passage of cells, while plasma may pass through the filtration matrix without inhibition. In some cases, the filtration matrix comprises a membrane that is particularly suitable for this application, as it combines large pore sizes at the top of the filter with small pore sizes at the bottom of the filter, which results in a very gentle treatment of the cells, preventing cell degradation during filtration.

In some cases, the devices disclosed herein comprise a separation material that moves, draws, pushes, or pulls the biological sample through the sample purifier, filter, and/or membrane. In some cases, the material is a wicking material. Examples of suitable separation materials for removing cells in the sample purifier include, but are not limited to, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, nitrocellulose, polycarbonate, polyethylene terephthalate, polyethylene, polypropylene, glass fiber, borosilicate, vinyl chloride, silver. Suitable separation materials may be characterized as preventing the passage of cells. In some cases, the separation material may prevent red blood cells from passing through. In some cases, the separator materialThe material is a hydrophobic filter, such as a glass fiber filter, a composite filter, such as a Cytosep (e.g., Ahlstrom Filtration, or Pall Specialty Materials, Port Washington, NY), or a hydrophilic filter, such as cellulose (e.g., Pall Specialty Materials). In some cases, commercially available kits may be used (e.g.,blood card serum separation kit, cat, abcs, Arrayit Corporation, Sunnyvale, CA) separates whole blood into red blood cells, white blood cells, and serum components for further processing according to the methods, systems, and kits disclosed herein.

In some cases, the sample purifier comprises at least one filter or at least one membrane characterized by at least one pore size. In some cases, the sample purifier comprises a plurality of filters and/or membranes, wherein at least a first filter or membrane has a pore size that is different from a pore size of a second filter or membrane. In some cases, at least one pore size of at least one filter/membrane is from about 0.05 microns to about 10 microns. In some cases, at least one pore size of at least one filter/membrane is from about 0.05 microns to about 8 microns. In some cases, at least one pore size of at least one filter/membrane is about 0.05 microns to about 6 microns. In some cases, at least one pore size of at least one filter/membrane is from about 0.05 microns to about 4 microns. In some cases, at least one pore size of at least one filter/membrane is from about 0.05 microns to about 2 microns. In some cases, at least one pore size of at least one filter/membrane is about 0.05 microns to about 1 micron.

Mild sample purifiers, such as those comprising a filtration matrix, vertical filters, wicking material, or a membrane with pores that do not allow passage of cells, are particularly suitable for analyzing cell-free nucleic acids. For example, prenatal application of cell-free fetal nucleic acid in maternal blood faces the additional challenge of analyzing cell-free fetal nucleic acid in the presence of cell-free maternal nucleic acid, which produces a greater background signal for the former. By way of non-limiting example, a maternal blood sample may contain about 500 to 750 genomic equivalents of total cell-free DNA (maternal and fetal) per milliliter of whole blood when the sample is obtained without cell lysis or other cell disruption caused by the sample collection method. The fetal fraction in blood sampled from a pregnant female may be about 10%, about 50 to 75 genome equivalents per ml. The process of obtaining cell-free nucleic acids typically involves obtaining plasma from blood. Without careful manipulation, the maternal leukocytes may be destroyed, releasing additional cellular nucleic acid into the sample, creating a large background noise to the fetal cell-free nucleic acid. Typical white blood cell counts are about 4 x 10 < SP > 6 </SP > to 10 x 10 < SP > 6 </SP > cells per ml of blood, and thus the available nuclear DNA is about 4,000 to 10,000 times greater than the total cell-free DNA (cfDNA). Thus, even if only a small fraction of the maternal leukocytes are destroyed, releasing nuclear DNA into the plasma, the fetal fraction will be greatly reduced. For example, 0.01% leukocyte degradation can reduce the fetal fraction from 10% to about 5%. The devices, systems, and kits disclosed herein are directed to reducing these background signals.

In some cases, the devices, systems, and kits disclosed herein comprise binding moieties for producing modified samples that have been depleted of cells, cell fragments, nucleic acids, or proteins that are not desired or of interest. In some cases, the devices, systems, and kits disclosed herein comprise binding moieties for reducing cells, cell fragments, nucleic acids, or proteins in a biological sample that are not needed or of interest. In some cases, the devices, systems, and kits disclosed herein comprise a binding moiety for producing a modified sample enriched for a target cell, a target cell fragment, a target nucleic acid, or a target protein.

In some cases, the devices, systems, and kits disclosed herein comprise a binding moiety capable of binding a nucleic acid, a protein, a peptide, a cell surface marker, or a microbubble surface marker. In some cases, the devices, systems, and kits disclosed herein comprise a binding moiety for capturing extracellular vesicles or extracellular microparticles in a biological sample. In some cases, the extracellular vesicles contain at least one of DNA and RNA. In some cases, the devices, systems, and kits disclosed herein comprise reagents or components for analyzing DNA or RNA contained in extracellular vesicles. In some cases, the binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a protein, a peptide, a small molecule, or a combination thereof.

In some cases, the devices, systems, and kits disclosed herein comprise a binding moiety capable of interacting with or capturing extracellular vesicles released by a cell. In some cases, the cell is a fetal cell. In some cases, the cell is a placental cell. Fetal cells or placental cells can circulate in a biological fluid (e.g., blood) of a female pregnant subject. In some cases, extracellular vesicles are released from organs, glands, or tissues. By way of non-limiting example, an organ, gland, or tissue may be diseased, aged, infected, or growing. Non-limiting examples of organs, glands and tissues are brain, liver, heart, kidney, colon, pancreas, muscle, fat, thyroid, prostate, breast tissue and bone marrow.

In some cases, the devices, systems, and kits disclosed herein are capable of capturing and discarding extracellular vesicles or extracellular microparticles from a maternal sample to enrich a fetal/placental nucleic acid sample. In some cases, the extracellular vesicles are derived from a fetus/placenta. In some cases, the extracellular vesicles are derived from fetal cells. In some cases, extracellular vesicles are released by fetal cells. In some cases, extracellular vesicles are released by placental cells. The placental cells can be trophoblast cells. In some cases, the devices, systems, and kits disclosed herein comprise a cell binding moiety for capturing placental-cultured platelets, which may contain fetal DNA or RNA fragments. These can be captured/enriched with antibodies or other methods (low speed centrifugation). In such cases, the fetal DNA or RNA fragments can be analyzed as described herein to determine or indicate chromosomal information (e.g., gender). Alternatively or additionally, the devices, systems, and kits disclosed herein comprise a binding moiety for capturing extracellular vesicles or extracellular microparticles in a biological sample from maternal cells.

In some cases, the binding moiety is attached to a solid support, wherein after the binding moiety has been contacted with the biological sample, the solid support can be separated from the rest of the biological sample, or the biological sample can be separated from the solid support. Non-limiting examples of solid supports include beads, nanoparticles, magnetic particles, chips, microchips, fiber strips, polymer strips, membranes, matrices, columns, plates, or combinations thereof.

The devices, systems, and kits disclosed herein may comprise a cell lysis reagent. Non-limiting examples of cell lysis reagents include detergents such as NP-40, sodium lauryl sulfate, and salt solutions containing ammonium, chloride, or potassium. The devices, systems, and kits disclosed herein can have a cell lysis component. The cell lysis component may be structural or mechanical and is capable of lysing cells. By way of non-limiting example, a cell lysis component can shear cells to release intracellular components, such as nucleic acids. In some cases, the devices, systems, and kits disclosed herein do not comprise a cell lysis reagent. Some of the devices, systems, and kits disclosed herein are directed to analyzing cell-free nucleic acids.

Nucleic acid amplification

In general, the devices, systems, and kits disclosed herein are capable of amplifying nucleic acids. In some cases, the nucleic acid comprises DNA. The DNA may be genomic. The DNA may be mitochondrial. In some cases, the nucleic acid comprises RNA. In some cases, the nucleic acid comprises cell-free DNA. In some cases, the nucleic acid comprises cell-free genomic DNA. In some cases, the devices, systems, and kits disclosed herein comprise a reverse transcriptase to produce complementary DNA (cDNA) from RNA in a biological sample disclosed herein, wherein the cDNA can be amplified and/or analyzed similarly to genomic DNA described herein. The RNA can include circulating cell-free RNA. The nucleic acid may be cell-free fetal nucleic acid.

In some cases, the devices, systems, kits, and methods disclosed herein comprise at least one nucleic acid amplification reagent or use thereof. Non-limiting examples of nucleic acid amplification reagents are polymerases, primers, nucleic acid amplification buffers and free nucleotides.

In some cases, the devices, systems, and kits disclosed herein are capable of isothermal amplification of nucleic acids.A non-limiting example of isothermal amplification is as follows: Loop mediated isothermal amplification (L AMP), Strand Displacement Amplification (SDA), Helicase Dependent Amplification (HDA), Nicking Enzyme Amplification (NEAR), and Recombinase Polymerase Amplification (RPA). accordingly, the devices, systems, and kits disclosed herein may contain reagents necessary to perform isothermal amplification.A non-limiting example of isothermal amplification reagents includes polymerase, Single-stranded DNA binding protein, and Strand Displacement polymerase.isothermal amplification using RPA (RPA) employs three core enzymes, namely recombinase, DNA binding protein, and recombinase, and oligonucleotide displacement polymerase, and the pairing of the oligonucleotide with the sequence of the oligonucleotide in (1) and the oligonucleotide displacement primer (2) is stabilized against the action of the DNA by the polymerase at room temperature (37 deg.C) and the use of the homologous primer for extension of the DNA by the polymerase at room temperature.

In some cases, the devices, systems, and kits disclosed herein are capable of amplifying nucleic acids at a single temperature. In some cases, the devices, systems, and kits disclosed herein may advantageously be operated at room temperature. In some cases, the devices, systems, and kits disclosed herein are capable of isothermal amplification of nucleic acids at a temperature of about 20 ℃ to about 65 ℃. In some cases, the devices, systems, and kits disclosed herein are capable of isothermal amplification of nucleic acids at about 23 ℃ to about 27 ℃. In some cases, the devices, systems, and kits disclosed herein are capable of amplifying nucleic acids at no more than two temperatures. In some cases, the devices, systems, and kits disclosed herein are capable of amplifying nucleic acids at no more than three temperatures. In some cases, the devices, systems, and kits disclosed herein require only initial heating of one reagent or component of the device, system, or kit.

In some cases, the devices, systems, kits, and methods disclosed herein comprise a hybridization probe with an abasic site, a fluorophore, and a quencher to monitor amplification. An endonuclease or exonuclease such as endonuclease IV or exonuclease III may be included to cleave the abasic site and release a quencher to allow fluorescence excitation. In some cases, the amplification product is detected or monitored by lateral flow by attaching a capture molecule (e.g., biotin) to one of the amplification primers and capturing with a 5' -antigen molecule (e.g., fluorescein derivative FAM) to label the hybrid primer to allow detection. As such, in some cases, the devices, systems, kits, and methods disclosed herein provide for the detection of nucleic acids and amplification products on a lateral flow device. Lateral flow devices are described herein.

In some cases, the devices, systems, and kits disclosed herein comprise at least one nucleic acid amplification reagent and at least one oligonucleotide primer capable of amplifying a first sequence in the genome and a second sequence in the genome, wherein the first sequence and the second sequence are similar, and wherein the first sequence is physically far enough from the second sequence that the first sequence is present on a first cell-free nucleic acid of the subject and the second sequence is present on a second cell-free nucleic acid of the subject. In some cases, the at least two sequences are immediately adjacent. In some cases, the at least two sequences are separated by at least one nucleotide. In some cases, the at least two sequences are separated by at least two nucleotides. In some cases, the at least two sequences are separated by at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or at least about 100 nucleotides. In some cases, the at least two sequences are at least about 50% identical. In some cases, the at least two sequences are at least about 60% identical, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% identical. In some cases, the first sequence and the second sequence are each at least 10 nucleotides in length. In some cases, the first sequence and the second sequence are each at least about 10, at least about 15, at least about 20, at least about 30, at least about 50, or at least about 100 nucleotides in length. In some cases, the first sequence and the second sequence are on the same chromosome. In some cases, the first sequence is on a first chromosome and the second sequence is on a second chromosome. In some cases, the first sequence and the second sequence are functionally linked. For example, all CpG sites in the promoter region of gene AOX1 show the same hypermethylation in prostate cancer and therefore these sites are functionally linked in that they carry functionally the same information but are located one or more nucleotides apart.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe or an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a region of interest or a portion thereof. In some cases, the region of interest is a region of the Y chromosome. In some cases, the region of interest is a region of the X chromosome. In some cases, the region of interest is a region of an autosome. In some cases, the region of interest or portion thereof comprises a repeat sequence as described herein, which is present more than once in the genome.

In some cases, the length of a region of interest disclosed herein is from about 10 nucleotides to about 1,000,000 nucleotides. In some cases, the region of interest is at least 10 nucleotides in length. In some cases, the region of interest is at least 100 nucleotides in length. In some cases, the region is at least 1000 nucleotides in length. In some cases, the region of interest is from about 10 nucleotides to about 500,000 nucleotides in length. In some cases, the region of interest is from about 10 nucleotides to about 300,000 nucleotides in length. In some cases, the region of interest is from about 100 nucleotides to about 1,000,000 nucleotides in length. In some cases, the region of interest is from about 100 nucleotides to about 500,000 nucleotides in length. In some cases, the region of interest is from about 100 nucleotides to about 300,000 base pairs in length. In some cases, the region of interest is from about 1000 nucleotides to about 1,000,000 nucleotides in length. In some cases, the region of interest is from about 1000 nucleotides to about 500,000 nucleotides in length. In some cases, the region of interest is from about 1000 nucleotides to about 300,000 nucleotides in length. In some cases, the region of interest is from about 10,000 nucleotides to about 1,000,000 nucleotides in length. In some cases, the region of interest is from about 10,000 nucleotides to about 500,000 nucleotides in length. In some cases, the region of interest is from about 10,000 nucleotides to about 300,000 nucleotides in length. In some cases, the region of interest is about 300,000 nucleotides in length.

In some cases, the sequence corresponding to the region of interest is at least about 5 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 8 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 10 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 15 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 20 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 50 nucleotides in length. In some cases, the sequence corresponding to the region of interest is at least about 100 nucleotides in length. In some cases, the sequence is from about 5 nucleotides to about 1000 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 1000 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 500 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 400 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 300 nucleotides in length. In some cases, the sequence is from about 50 nucleotides to about 1000 nucleotides in length. In some cases, the sequence is from about 50 nucleotides to about 500 nucleotides in length.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a region of interest disclosed herein. In some cases, the sub-region is represented by a sequence that exists more than once in the region of interest. In some cases, the sub-region is about 10 to about 1000 nucleotides in length. In some cases, the sub-region is about 50 to about 500 nucleotides in length. In some cases, the sub-region is about 50 to about 250 nucleotides in length. In some cases, the sub-region is about 50 to about 150 nucleotides in length. In some cases, the length of the sub-region is about 100 nucleotides.

In some cases, the devices, systems, and kits disclosed herein comprise at least one oligonucleotide primer, wherein the oligonucleotide primer has a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the devices, systems, and kits disclosed herein comprise a pair of oligonucleotide primers, wherein the pair of oligonucleotide primers has a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the devices, systems, and kits disclosed herein comprise at least one oligonucleotide primer, wherein the oligonucleotide primer comprises a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the devices, systems, and kits disclosed herein comprise a pair of oligonucleotide primers, wherein the pair of oligonucleotide primers comprises a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the devices, systems, and kits disclosed herein comprise at least one oligonucleotide primer, wherein the oligonucleotide primer consists of a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the devices, systems, and kits disclosed herein comprise an oligonucleotide primer pair, wherein the oligonucleotide primer pair consists of a sequence that is complementary to or corresponds to a Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 75% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 80% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 85% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 80% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 90% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 95% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is at least 97% identical to the wild-type human Y chromosome sequence. In some cases, the sequence complementary to or corresponding to the Y chromosome sequence is 100% identical to the wild-type human Y chromosome sequence.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome region or a portion thereof, wherein the portion thereof has a given length. In some cases, the portion thereof is from about 10 nucleotides to about 100 nucleotides in length. In some cases, the portion thereof is from about 100 nucleotides to about 1000 nucleotides in length. In some cases, the portion thereof is from about 1000 nucleotides to about 10,000 nucleotides in length. In some cases, the portion thereof is from about 10,000 nucleotides to about 100,000 nucleotides in length.

In some cases, the region of interest is a Y chromosome region or portion thereof comprising a sequence that is present more than once on the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20500000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 20500000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 20250000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20250000 and position 20500000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20500000 and position 20750000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20750000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20080000 and position 20400000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20082000 and position 20351000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20082183 and position 20350897 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion. In some cases, the correspondence is 100% identical. In some cases, the correspondence is at least 99% identical. In some cases, the correspondence is at least 98% identical. In some cases, the correspondence is at least 95% identical. In some cases, the correspondence is at least 90% identical.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion between the start position 20350799 and the stop position 20350897 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion between the start position 20349236 and the stop position 20349318 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion between the start position 20350231 and the stop position 20350323 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion between the start position 20350601 and the stop position 20350699 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a sub-region of the Y chromosome between start position 20082183 and stop position 20082281 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 20082183 and the end position 20082281 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 20082183 and the end position 20082281 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the starting position 20082183 and the ending position 20082281 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the starting position 20082183 and the ending position 20082281 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the starting position 20082183 and the ending position 20082281 of the Y chromosome.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion between the start position 56673250 and the stop position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion, wherein the sequence is selected from SEQ ID nos. 1-5 shown in table 1. In some cases, the sequence is at least 60% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 65% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 70% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 75% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 80% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 85% identical to a sequence selected from SEQ id nos. 1-5. In some cases, the sequence is at least 90% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 95% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 98% identical to a sequence selected from SEQ ID No. 1-5. In some cases, the sequence is at least 99% identical to a sequence selected from seq id No. 1-5. In some cases, the correspondence is 100% identical. In some cases, the sequence comprises at least 10 contiguous nucleotides of SEQ ID No. 1-5. In some cases, the sequence comprises at least 15 contiguous nucleotides of SEQ ID No. 1-5. In some cases, the sequence comprises at least 20 contiguous nucleotides of SEQ ID No. 1-5. In some cases, the sequence comprises at least 25 contiguous nucleotides of SEQ ID No. 1-5. In some cases, the sequence comprises at least 50 contiguous nucleotides of SEQ ID No. 1-5.

TABLE 1 sequences of the Y chromosome subregions

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion, wherein the sequence is selected from SEQ ID nos. 30-34 shown in table 3. In some cases, the sequence is at least 60% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 65% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 70% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 75% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 80% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 85% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 90% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 95% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 98% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the sequence is at least 99% identical to a sequence selected from SEQ ID No. 30-34. In some cases, the correspondence is 100% identical. In some cases, the sequence comprises at least 10 contiguous nucleotides of SEQ ID No. 30-34. In some cases, the sequence comprises at least 15 contiguous nucleotides of SEQ ID No. 30-34. In some cases, the sequence comprises at least 20 contiguous nucleotides of SEQ ID No. 30-34. In some cases, the sequence comprises at least 25 contiguous nucleotides of SEQ ID No. 30-34. In some cases, the sequence comprises at least 50 contiguous nucleotides of SEQ ID No. 30-34. Example 3 describes the results of an assay to analyze the Y chromosome subregion having a sequence selected from SEQ ID No. 30-34.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence between chrY:56672250 and chrY:56772489 (according to genomic version 38). In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence between chrY:56673250 and chrY: 56771489. (construct 38 from the genome). Example 4 shows the results using primer pairs (shown in table 5) that amplify such sequences. The primer pair may be selected from primers represented by two sequences selected from: 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127, 125 and 128, 129 and 130, 131 and 132, 137 and 134, 135 and 136, 137 and 138, and 140. In some cases, the primer pair is represented by a sequence that is at least 80% identical to a sense primer in table 5 and at least 90% identical to an antisense primer in table 5. In some cases, the primer pair is represented by a sequence that is at least 90% identical to a sense primer in table 5 and at least 90% identical to an antisense primer in table 5.

In some cases, the devices, systems, and kits disclosed herein comprise at least one of an oligonucleotide probe and an oligonucleotide primer capable of annealing to a cell-free nucleic acid strand, wherein the cell-free nucleic acid comprises a sequence corresponding to a Y chromosome subregion, wherein the sequence is selected from SEQ ID No. 141-. In some cases, the sequence is at least 60% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 65% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 70% identical to a sequence selected from the group consisting of SEQ ID NO. 141 and 192. In some cases, the sequence is at least 75% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 85% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 90% identical to a sequence selected from SEQ ID NO. 141-192. In some cases, the sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO. 141-192. In some cases, the sequence is at least 99% identical to a sequence selected from the group consisting of SEQ ID NO. 141 and 192. In some cases, the correspondence is 100% identical. In some cases, the sequence comprises at least 10 contiguous nucleotides of SEQ ID NO. 141-192. In some cases, the sequence comprises at least 15 contiguous nucleotides of SEQ ID NO. 141-192. In some cases, the sequence comprises at least 20 contiguous nucleotides of SEQ ID NO. 141-192. In some cases, the sequence comprises at least 25 contiguous nucleotides of SEQ ID NO. 141-192. In some cases, the sequence comprises at least 50 contiguous nucleotides of SEQ ID NO. 141-192. Example 4 describes the results of an assay to analyze the sub-region of the Y chromosome having a sequence selected from the group consisting of SEQ ID NO. 141 and 192.

Nucleic acid sequencing

In some cases, the devices, systems, and kits disclosed herein comprise a nucleic acid sequencer. In some cases, the devices, systems, and kits disclosed herein are configured to amplify nucleic acids and sequence the resulting amplified nucleic acids. In some cases, the devices, systems, and kits disclosed herein are configured to sequence nucleic acids without amplifying the nucleic acids. In some cases, the devices, systems, and kits disclosed herein comprise a nucleic acid sequencer, but no nucleic acid amplification reagents or nucleic acid amplification components. In some cases, the nucleic acid sequencer comprises a signal detector that detects a signal reflective of successful amplification or unsuccessful amplification. In some cases, the nucleic acid sequencer is a signal detector. In some cases, the signal detector comprises a nucleic acid sequencer.

In some cases, the nucleic acid sequencer has a communication connection with electronics that analyze sequencing reads from the nucleic acid sequencer. In some cases, the communication connection is hardwired. In some cases, the communication connection is wireless. For example, a cell phone application or computer software such as those disclosed herein may receive sequencing reads and display or report genetic information about the sample (e.g., presence of disease/infection, response to medication, gender of fetus) based on the sequencing reads.

In some cases, the nucleic acid sequencer comprises a nanopore sequencer. In some cases, the nanopore sequencer comprises a nanopore. In some cases, a nanopore sequencer comprises a membrane and a solution that generates a current across the membrane and drives a charged molecule (e.g., a nucleic acid) to move through the nanopore. In some cases, the nanopore sequencer comprises a transmembrane protein, a portion thereof, or a modification thereof. In some cases, the transmembrane protein is a bacterial protein. In some cases, the transmembrane protein is not a bacterial protein. In some cases, the nanopore is synthetic. In some cases, the nanopore performs solid state nanopore sequencing. In some cases, nanopore sequencers are described as pocket-sized, portable, or substantially cell-phone sized. In some cases, the nanopore sequencer is configured to sequence at least one of RNA and DNA. Non-limiting examples of Nanopore sequencing devices include Oxford Nanopore Technologies MinION and SmidgION Nanopore sequencing USB devices. Both devices are small enough to be hand-held. Nanopore sequencing devices and components are further described in reviews by Howorta (Nat nanotechnol.2017, 7/6; 12(7): 619-. Other non-limiting examples of nanopore sequencing devices are provided by Electronic Biosciences, Two Pore Guys, Stratos, and Agilent (originally from Genia).

In some cases, the devices, systems, and kits disclosed herein comprise reagents and components necessary for bisulfite sequencing to detect epigenetic modifications. For example, long regions with many methylation markers can be fragmented. Here, each fragment carrying a methylation marker can be an independent signal. The combination of signals from all fragments is sufficient to obtain useful genetic information.

Capture and detection

In some cases, the devices, systems, and kits disclosed herein comprise at least one of a capture assembly, a signal detector, and a detection reagent for detecting nucleic acids in a biological sample. In some cases, the capture assembly and the signal detector are integrated. In some cases, the capture assembly comprises a solid support. In some cases, the solid support comprises a bead, chip, strip, membrane, matrix, column, plate, or a combination thereof. In some cases, the capture assembly comprises a binding moiety disclosed herein.

In some cases, the devices, systems, and kits disclosed herein comprise at least one probe for a nucleic acid having a sequence of interest. In some cases, the sequence of interest is specific for the Y chromosome. In some cases, the devices, systems, and kits disclosed herein comprise at least one probe for a paternal genetic sequence that is not present in maternal DNA. In some cases, the devices, systems, and kits disclosed herein comprise at least one probe for a single nucleotide polymorphism inherited by the father. In some cases, the devices, systems, and kits disclosed herein comprise at least one probe directed to an epigenetically modified region of a chromosome or fragment thereof. In some cases, the epigenetic modification of the epigenetic modified region of the chromosome is indicative of gender or a marker of gender. In some cases, the chromosome is the Y chromosome. In some cases, the chromosome is an X chromosome. In some cases, the chromosome is an autosome. In some cases, the probe comprises a peptide, an antibody, an antigen-binding antibody fragment, a nucleic acid, or a small molecule.

In some cases, the capture assembly comprises a binding moiety described herein. In some cases, the binding moiety is present in a lateral flow assay. In some cases, the binding moiety is added to the sample prior to adding the sample to the lateral flow assay. In some cases, the binding moiety comprises a signal molecule. In some cases, the binding moiety is physically associated with the signal molecule. In some cases, the binding moiety is capable of physically associating with a signal molecule. In some cases, the binding moiety is linked to a signal molecule. Non-limiting examples of signal molecules include gold particles, fluorescent particles, luminescent particles, and dye molecules. In some cases, the capture component comprises a binding moiety capable of interacting with an amplification product described herein. In some cases, the capture component comprises a binding moiety capable of interacting with a tag on an amplification product described herein.

In some cases, the devices, systems, and kits disclosed herein comprise a detection system. In some cases, the detection system includes a signal detector. Non-limiting examples of signal detectors include fluorescence readers, colorimeters, sensors, wires, circuits, receivers. In some cases, the detection system comprises a detection reagent. Non-limiting examples of detection reagents include fluorophores, chemicals, nanoparticles, antibodies, and nucleic acid probes. In some cases, the detection system comprises a pH sensor and a complementary metal oxide semiconductor, which can be used to detect a change in pH. In some cases, the generation of amplification products by the devices, systems, kits, or methods disclosed herein changes pH, thereby indicating gender.

In some cases, the detection system includes a signal detector. In some cases, the signal detector is a photodetector that detects photons. In some cases, the signal detector detects fluorescence. In some cases, the signal detector detects a chemical or compound. In some cases, the signal detector detects a chemical released when an amplification product is produced. In some cases, the signal detector detects a chemical released upon addition of the amplification product to the detection system. In some cases, the signal detector detects compounds produced when the amplification product is produced. In some cases, the signal detector detects a compound produced when the amplification product is added to the detection system.

In some cases, the signal detector detects an electrical signal. In some cases, the signal detector comprises an electrode. In some cases, the signal detector includes a circuit, a current, or a current generator. In some cases, the circuit or current is provided by a gradient of two or more solutions or polymers. In some cases, the circuit or current is provided by an energy source (e.g., a battery, a cord of an electrical outlet). In some cases, a nucleic acid, amplification product, chemical, or compound disclosed herein provides an electrical signal by an interference current, and a signal detector detects the electrical signal. In some cases, the signal detector detects light. In some cases, the signal detector comprises a light sensor. In some cases, the signal detector comprises a camera. In some cases, the signal detector comprises a cell phone camera or a component thereof.

In some cases, the signal detector comprises a nanowire that detects the charge of different bases in the nucleic acid. In some cases, the nanowires have a diameter of about 1nm to about 99 nm. In some cases, the nanowires have a diameter of about 1nm to about 999 nm. In some cases, the nanowires comprise inorganic molecules, such as nickel, platinum, silicon, gold, zinc, graphene, or titanium. In some cases, the nanowires comprise organic molecules (e.g., nucleotides).

In some cases, the detection system comprises an assay assembly, wherein the assay assembly is capable of detecting a target analyte (e.g., a nucleic acid amplification product). In some cases, the assay assembly comprises a lateral flow strip, also referred to herein and in the art as a lateral flow assay, lateral flow test, or lateral flow device. In some cases, lateral flow assays provide a rapid, inexpensive, and technically simple method to detect the amplification products disclosed herein. In general, the lateral flow assays disclosed herein comprise a porous material or matrix that transports a fluid, and a detector that detects the amplification products when present. The porous material may comprise porous paper, polymeric structures, sintered polymers, or combinations thereof. In some cases, the lateral flow assay transports a biological fluid or a portion thereof (e.g., plasma of a blood sample). In some cases, the lateral flow assay transports a solution containing a biological fluid or portion thereof. For example, the method may include adding a solution to the biological fluid before or during adding the sample to the device or system. The solution may comprise a salt, polymer, or any other component that facilitates transport of the sample and/or amplification products by the lateral flow assay. In some cases, the nucleic acid is amplified after traveling through the lateral flow strip.

In some cases, the apparatus, detection system, comprises a lateral flow device, wherein the lateral flow device comprises a plurality of zones or regions, wherein each desired function may be present in a separate zone or region. Typically, in a lateral flow device, a liquid sample containing a target analyte, such as a bodily fluid sample as described herein, moves through a section or region of the lateral flow device with or without the aid of external forces. In some cases, the target analyte without the aid of external force to move, for example, by capillary action. In some cases, the target analyte moves with the assistance of an external force, e.g., the promotion of capillary action by movement of a lateral flow device. Movement may include any movement caused by an external input, such as shaking, rotation, centrifugation, application of an electric or magnetic field, application of an active pump, application of a vacuum, or rocking of the lateral flow device.

In some cases, the lateral flow device is a lateral flow test strip comprising regions or sections located laterally, e.g., behind or in front of each other. Typically, lateral flow test strips allow access to the functional regions or sections from each side (e.g., above and below) of the test strip due to the exposure of a larger surface area of each functional region or section. This facilitates the addition of reagents, including those used in sample purification or target analyte amplification, detection and/or assay.

Lateral Flow test strip assay formats are generally described by, for example, shara et al, (2015) Biosensors 5: 577-.

In some cases, the lateral flow test strip uses a sandwich format, a competition format, or a multiplex detection format to detect the target analyte in the test sample. In conventional sandwich assay formats, the detected signal is directly proportional to the amount of target analyte present in the sample, and thus an increase in the amount of target analyte results in an increase in signal intensity. In conventional competitive assay formats, the detected signal is inversely related to the amount of analyte present, and increasing the amount of analyte results in a decrease in signal intensity.

In a lateral flow sandwich format, the test sample is typically applied to a sample application pad at one end of the test strip. The applied test sample flows from the sample application pad, through the test strip, to a conjugate pad located adjacent to the sample application pad, wherein the conjugate pad is downstream in the direction of sample flow. In some cases, the conjugate pad comprises a labeled, reversibly immobilized probe, e.g., an antibody or aptamer labeled with, e.g., a dye, enzyme, or nanoparticle. If the target analyte is present in the test sample, a labeled probe-target analyte complex is formed. The complex then flows to a first test area or zone (e.g., test line) containing immobilized second probes specific for the target analyte, thereby capturing any labeled probe-target analyte complexes. In some cases, the intensity or magnitude of the signal (e.g., color) at the first test area or segment is used to indicate the presence or absence, amount, or both presence and amount of the target analyte in the test sample. The second test region or zone may comprise a third probe bound to an excess of labelled probe. If the applied test sample contains the target analyte, little or no excess labeled probes will be present on the test strip after the target analyte is captured by the labeled probes on the conjugate pad. Thus, the second test area or zone will not bind any labeled probes and little or no signal (e.g., color) is expected to be observed in the second test area or zone. Thus, the absence of a signal at the second test area or zone may provide assurance that the signal observed in the first test area or zone is due to the presence of the target analyte.

In some cases, the devices and systems disclosed herein comprise sandwich assays. In some cases, a sandwich assay is configured to receive a biological sample disclosed herein and retain sample components (e.g., nucleic acids, cells, microparticles). In some cases, the sandwich assay is configured to receive a flowing solution that washes non-nucleic acid components (e.g., proteins, cells, microparticles) of the biological sample, leaving nucleic acids of the biological sample. In some cases, sandwich assays comprise a membrane bound to nucleic acids to help retain the nucleic acids when a flow solution is applied. Non-limiting examples of membranes that bind nucleic acids include chitosan-modified nitrocellulose.

Similarly, in a lateral flow competition format, a test sample is applied to a sample application pad at one end of the test strip, and the target analyte binds to the labeled probes to form probe-target analyte complexes in a conjugate pad downstream of the sample application pad. In a competitive format, the first test zone or segment typically comprises the target analyte or an analog of the target analyte. The target analyte of the first test zone or segment binds to any free labeled probes in the conjugate pad that do not bind to the test analyte. Thus, when the target analyte is not present in the applied test sample, a higher amount of signal is observed in the first test area or zone than when the target analyte is present. The second test zone or segment comprises probes that specifically bind to the probe-target analyte complex. The amount of signal observed in this second test zone or segment is higher when the target analyte is present in the applied test sample.

In a lateral flow test strip multiplex detection format, more than one target analyte is detected using the test strip by using additional test zones or segments containing, for example, probes specific for each target analyte.

In some cases, the lateral flow device is a layered lateral flow device comprising regions or sections that are present in layers that are located in the middle (e.g., above or below each other). In some cases, there are one or more regions or zones in a given layer. In some cases, each region or section is present in a separate layer. In some cases, a layer includes multiple regions or sections. In some cases, the layers are laminated. In a layered lateral flow device, diffusion-controlled and concentration gradient-guided processes are possible driving forces. For example, a multi-layer analytical element for performing a fluorescence assay or a quantitative fluorescence analysis on an analyte contained in a sample liquid is described in EP0097952 "multi-layer analytical element", which is incorporated herein by reference.

The lateral flow device may comprise one or more functional regions or zones. In some cases, the test assembly contains 1 to 20 functional areas or segments. In some cases, the functional regions or zones comprise at least one sample purification region or zone, at least one target analyte amplification region or zone, at least one target analyte detection region or zone, and at least one target analyte assay region or zone.

In some cases, the target analyte is a nucleic acid sequence and the lateral flow device is a nucleic acid lateral flow assay. In some cases, the devices, systems, and kits disclosed herein comprise a nucleic acid lateral flow assay, wherein the nucleic acid lateral flow assay comprises a nucleic acid amplification function. In some cases, amplification of the target nucleic acid by the nucleic acid amplification function is performed prior to or simultaneously with detection of the amplified nucleic acid species. In some cases, the detection comprises one or more of a qualitative, semi-quantitative, or quantitative determination of the presence of the target analyte.

In some cases, the devices, systems, and kits disclosed herein comprise an assay component in which a target nucleic acid analyte is amplified in a lateral flow test strip to generate labeled amplification products, or amplification products that can be labeled after amplification. In some cases, the label is present on, or subsequently conjugated to, one or more amplification primers after amplification. In some cases, at least one target nucleic acid amplification product is detected on the lateral flow test strip. For example, one or more regions or segments on the lateral flow test strip can comprise a probe specific for a target nucleic acid amplification product.

In some cases, the devices, systems, and kits disclosed herein comprise a detector, wherein the detector comprises a graphene biosensor. Graphene biosensors are described, for example, by Afsahi et al in an article entitled "Novel graphene-based Biosensor for early detection of Zika virus infection, Biosensor and Biosensor electronics," (2018)100: 85-88.

In some cases, the detectors disclosed herein include nanopores, nanosensors, or nanoswitches. For example, a detector that measures current perturbations corresponding to a particular nucleotide may be capable of nanopore sequencing, which is a method of transporting a nucleic acid through a nanopore based on transmembrane current. The nanoswitches or nanosensors undergo a structural change upon exposure to a detectable signal. See, for example, Koussa et al, "DNA nanoswitches: A quantitative platform for gel-based biomolecular interaction analysis," (2015) Nature methods,12(2): 123-.

In some cases, the detector comprises a rapid multiplex biomarker assay, wherein probes for the analyte of interest are generated on a chip for real-time detection. Thus, no labels, tags or reporters are required. Binding of analyte to these probes causes a change in refractive index corresponding to the analyte concentration. All steps may be automated. Incubation may not be necessary. Results can be obtained in less than one hour (e.g., 10-30 minutes). A non-limiting example of such a detector is the Genbyte Maverick detection System.

Additional testing

In some cases, the devices, systems, and kits disclosed herein comprise additional features, reagents, tests, or assays for detecting or analyzing biological components other than nucleic acids. By way of non-limiting example, the biological component may be selected from the group consisting of proteins, peptides, lipids, fatty acids, sterols, carbohydrates, viral components, microbial components, and combinations thereof. These additional assays may be capable of detecting or analyzing biological components in small volumes or sample sizes as disclosed herein and throughout. The additional test may comprise an agent capable of interacting with the biological component of interest. Non-limiting examples of such agents include antibodies, peptides, oligonucleotides, aptamers, and small molecules, and combinations thereof. The reagent may comprise a detectable label. The reagent may be capable of interacting with a detectable label. The reagent may be capable of providing a detectable signal.

For example, an additional test may comprise providing reagents or components for performing immuno-PCR (IPCR). IPCR is a method in which a first antibody directed against a protein of interest is immobilized and exposed to a sample, if the sample contains a protein of interest, it will be captured by the first antibody.

In some cases, the devices, systems, and kits disclosed herein comprise additional tests or assays in addition to the assay for the nucleic acid corresponding to the Y chromosome. In some cases, the methods disclosed herein comprise testing the biological sample in addition to testing for the presence of the Y chromosome (sex test). In some cases, the methods disclosed herein comprise characterizing the biological sample in addition to testing for the presence of the Y chromosome. In some cases, the devices, systems, and kits disclosed herein comprise a test for a protein or peptide. In some cases, the protein is a hormone. In some cases, the methods disclosed herein comprise testing, determining, or quantifying a protein. In some cases, the devices, systems, and kits disclosed herein comprise assays for the presence or amount of nucleic acids and the presence or amount of proteins or peptides. In some cases, the additional test is a test for gestational age. In some cases, testing for gestational age ensures that gender testing is performed at gestational age where accurate gender detection can be made. In some cases, the additional test is a pregnancy test. In some cases, a pregnancy test confirms that a female is a subject if gender and/or gestational age cannot be detected or discerned by a device, system, or kit disclosed herein.

In some cases, the devices, systems, and kits disclosed herein comprise a pregnancy test for indicating, determining, or verifying a pregnancy in a female subject. In some cases, the pregnancy test comprises a reagent or component for measuring a pregnancy-associated factor. By way of non-limiting example, the pregnancy-associated factor may be human chorionic gonadotropin (hCG) and an agent or module directed against hCG comprising an anti-hCG antibody. Also by way of non-limiting example, the pregnancy-associated factor may be an hCG transcript and the reagent or component for measuring the hCG transcript is an oligonucleotide probe or primer that hybridizes to the hCG transcript. In some cases, the pregnancy-associated factor is heat shock protein 10kDa protein 1, also known as Early Pregnancy Factor (EPF).

In some cases, the devices, systems, and kits disclosed herein are capable of communicating the age of the fetus. For example, a signal may be generated from the device or system, wherein the level of the signal corresponds to the amount of hCG in a sample from the subject. The level or intensity of this signal can be translated or otherwise expressed (equivocate) into a number indicative of the amount of hCG in the sample. The amount of hCG may be indicative of the approximate age of the fetus.

In some cases, the devices, systems, and kits disclosed herein provide an indication or verification of pregnancy, an indication or verification of gestational age, and an indication or verification of gender. In some cases, the devices, systems, and kits disclosed herein provide an indication of pregnancy, gestational age, and/or gender with at least about 90% confidence (e.g., in 90% of cases, the indication is accurate). In some cases, the devices, systems, and kits disclosed herein provide an indication of pregnancy, gestational age, and/or gender with a confidence of at least about 95%. In some cases, the devices, systems, and kits disclosed herein provide an indication of pregnancy, gestational age, and/or gender with a confidence of at least about 99%.

Performance parameter

In some cases, the devices, systems, and kits disclosed herein are operable at one or more temperatures. In some cases, it is desirable to change the temperature of the components or reagents of the device system or kit in order for the device system or kit to be operational. Generally, devices, systems, and kits are considered operable when they provide information (e.g., gender, infection, contamination) conveyed by a biomarker (e.g., RNA/DNA, peptide) in a biological sample. In some cases, the device, system, kit, components thereof, or reagents thereof are obtained in the ordinary home at a temperature at which they are operable. By way of non-limiting example, the temperatures obtained in a typical household may be provided by room temperature, refrigerator, freezer, microwave, oven, hot/cold water bath, or oven.

In some cases, the devices, systems, kits, components thereof, or reagents thereof described herein are operable at a single temperature. In some cases, the devices, systems, kits, components thereof, or reagents thereof described herein require only a single temperature to be operable. In some cases, the devices, systems, kits, components thereof, or reagents thereof described herein require only two temperatures to be operable. In some cases, the devices, systems, kits, components thereof, or reagents thereof described herein require only three temperatures to be operable.

In some cases, the temperature at which the device, system, kit, components thereof, or reagents thereof are operable is within a temperature range or at least one temperature that falls within a temperature range. In some cases, the temperature ranges from about-50 ℃ to about 100 ℃. In some cases, the temperature ranges from about-50 ℃ to about 90 ℃. In some cases, the temperature ranges from about-50 ℃ to about 80 ℃. In some cases, the temperature ranges from about-50 ℃ to about 70 ℃. In some cases, the temperature ranges from about-50 ℃ to about 60 ℃. In some cases, the temperature ranges from about-50 ℃ to about 50 ℃. In some cases, the temperature ranges from about-50 ℃ to about 40 ℃. In some cases, the temperature ranges from about-50 ℃ to about 30 ℃. In some cases, the temperature ranges from about-50 ℃ to about 20 ℃. In some cases, the temperature ranges from about-50 ℃ to about 10 ℃. In some cases, the temperature ranges from about 0 ℃ to about 100 ℃. In some cases, the temperature ranges from about 0 ℃ to about 90 ℃. In some cases, the temperature ranges from about 0 ℃ to about 80 ℃. In some cases, the temperature ranges from about 0 ℃ to about 70 ℃. In some cases, the temperature range is from about 0 ℃ to about 60 ℃. In some cases, the temperature ranges from about 0 ℃ to about 50 ℃. In some cases, the temperature range is from about 0 ℃ to about 40 ℃. In some cases, the temperature ranges from about 0 ℃ to about 30 ℃. In some cases, the temperature ranges from about 0 ℃ to about 20 ℃. In some cases, the temperature range is from about 0 ℃ to about 10 ℃. In some cases, the temperature ranges from about 15 ℃ to about 100 ℃. In some cases, the temperature ranges from about 15 ℃ to about 90 ℃. In some cases, the temperature ranges from about 15 ℃ to about 80 ℃. In some cases, the temperature ranges from about 15 ℃ to about 70 ℃. In some cases, the temperature range is from about 15 ℃ to about 60 ℃. In some cases, the temperature ranges from about 15 ℃ to about 50 ℃. In some cases, the temperature range is from about 15 ℃ to about 40 ℃. In some cases, the temperature ranges from about 15 ℃ to about 30 ℃. In some cases, the temperature range is from about 10 ℃ to about 30 ℃. In some cases, the devices, systems, kits disclosed herein, including all of their components and all of their reagents, are fully operational at room temperature without the need for cooling, freezing, or heating.

In some cases, the devices, systems, and kits disclosed herein comprise a heating device or a cooling device to allow a user to obtain at least one temperature or temperature range. Non-limiting examples of heating and cooling devices are pouches or bags that can be cooled in a freezer or freezer, or heated with a microwave oven, oven or stove top. In some cases, a heating or cooling device is plugged into an electrical outlet and then applied to the device or its components disclosed herein, thereby transferring heat to or cooling the device or its components. Another non-limiting example of a heating device is a wire or coil that passes through the device or a portion thereof. The wires or coils may be activated by an external (e.g., solar, socket) or internal (e.g., battery) power source to transfer heat to the device or portions thereof. In some cases, the devices, systems, kits disclosed herein include a thermometer or temperature indicator to assist a user in determining that a suitable temperature or temperature range has been obtained for the device, system, or component thereof. Alternatively or additionally, the user uses a device (e.g., thermometer, cell phone, etc.) in a typical home environment to assess temperature.

In some cases, the devices, systems, and kits disclosed herein detect components of a biological sample or a product thereof (e.g., amplification product, conjugation product, binding product) over a time frame in which the biological sample is received. In some cases, detection occurs by a signal molecule described herein. In some cases, the time ranges from about 1 second to about 1 minute. In some cases, the time ranges from about 10 seconds to about 1 minute. In some cases, the time ranges from about 20 seconds to about 1 minute. In some cases, the time ranges from about 30 seconds to about 1 minute. In some cases, the time ranges from about 10 seconds to about 2 minutes. In some cases, the time ranges from about 10 seconds to about 3 minutes. In some cases, the time ranges from about 10 seconds to about 5 minutes. In some cases, the time ranges from about 10 seconds to about 10 minutes. In some cases, the time ranges from about 10 seconds to about 15 minutes. In some cases, the time ranges from about 10 seconds to about 20 minutes. In some cases, the time ranges from about 30 seconds to about 2 minutes. In some cases, the time ranges from about 30 seconds to about 5 minutes. In some cases, the time ranges from about 30 seconds to about 10 minutes. In some cases, the time ranges from about 30 seconds to about 15 minutes. In some cases, the time ranges from about 30 seconds to about 20 minutes. In some cases, the time ranges from about 30 seconds to about 30 minutes. In some cases, the time ranges from about 1 minute to about 2 minutes. In some cases, the time ranges from about 1 minute to about 5 minutes. In some cases, the time ranges from about 1 minute to about 10 minutes. In some cases, the time ranges from about 1 minute to about 20 minutes. In some cases, the time ranges from about 1 minute to about 30 minutes. In some cases, the time ranges from about 5 minutes to about 10 minutes. In some cases, the time ranges from about 5 minutes to about 15 minutes. In some cases, the time ranges from about 5 minutes to about 20 minutes. In some cases, the time ranges from about 5 minutes to about 30 minutes. In some cases, the time ranges from about 5 minutes to about 60 minutes.

In some cases, the devices, systems, and kits disclosed herein detect components of a biological sample or product thereof (e.g., amplification product, conjugation product, binding product) in less than a given amount of time. In some cases, the devices, systems, and kits disclosed herein provide for analysis of a component of a biological sample or product thereof in less than a given amount of time. In some cases, the amount of time is less than 1 minute. In some cases, the amount of time is less than 5 minutes. In some cases, the amount of time is less than 10 minutes. In some cases, the amount of time is less than 15 minutes. In some cases, the amount of time is less than 20 minutes. In some cases, the amount of time is less than 30 minutes. In some cases, the amount of time is less than 60 minutes. In some cases, the amount of time is less than 2 hours. In some cases, the amount of time is less than 8 hours.

Communication and information storage

Preferably, the devices, systems, and kits disclosed herein comprise a communication link or interface such that the obtained genetic information can be shared with others not physically present. The communication connection or interface may also allow input from other sources. In some cases, the devices, systems, and kits disclosed herein comprise an interface for receiving information based on the obtained genetic information. The interface or communication connection may also receive non-genetic information from the user (e.g., medical history, medical condition, age, weight, etc.). The interface or communication connection may also receive information provided by other people or things than the user. By way of non-limiting example, this includes network-based information, information from healthcare practitioners, and information from insurance companies. For example, the devices, systems, and kits disclosed herein may comprise or communicate with an artificial intelligence interface that sells gender-related or gender-specific products to pregnant subjects based on gender results of the test. In some cases, the devices, systems, and kits disclosed herein comprise an information storage unit, e.g., a computer chip. In some cases, the devices, systems, and kits disclosed herein comprise a means for securely storing genetic information. For example, the devices, systems, and kits disclosed herein may contain a data chip or connection (wired or wireless) to a hard disk, server, database, or cloud.

In some cases, the devices, systems, and kits disclosed herein are capable of communicating information about a biomarker in a biological sample to a communication device. In some cases, the communication device is connected to the internet. In some cases, the communication device is not connected to the internet. In some cases, the devices, systems, and kits disclosed herein are capable of communicating information about biomarkers in a biological sample to the internet via a communication device. Non-limiting examples of communication devices are cell phones, electronic notebooks, and computers.

In some cases, the devices, systems, and kits disclosed herein are capable of identifying and storing intermediate results of the respective tests. The intermediate results may indicate which test parameters (e.g., analyte, reagent, label, method, or device component) are useful or accurate. This information can be useful feedback for developing a team of tests or assays using the devices, systems, and kits disclosed herein. The team receiving this feedback may select new, better or optimal parameters based on this information, or may again ensure that they have selected the optimal parameters.

In some cases, the devices, systems, and kits disclosed herein comprise a communication connection or communication interface. In some embodiments, the communication interface provides a wired interface. In further embodiments, the wired communication interface utilizes Universal Serial Bus (USB) (including mini-USB, micro-USB, USB Type a, USB Type B, and USB Type C), IEEE1394(FireWire), Thunderbolt, ethernet, and fiber optic interconnects.

In further embodiments, the wireless communication interface utilizes a wireless communication protocol, such as infrared, Near Field Communication (NFC) (including RFID), Bluetooth Low energy (B L E), ZigBee, ANT, IEEE802.11(Wi-Fi), wireless local area network (W L AN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (worldwide interoperability for microwave Access (WiMAX)), or 3G/4G/L TE/5G cellular communication methods.

In some embodiments, the devices, systems, kits, and methods described herein include digital processing devices or uses thereof. In further embodiments, the digital processing device includes one or more hardware Central Processing Units (CPUs) or general purpose graphics processing units (gpgpgpgpu) that perform device functions. In further embodiments, the digital processing device further comprises an operating system configured to execute the executable instructions. In some embodiments, the digital processing device includes a communication interface (e.g., a network adapter) for communicating with one or more peripheral devices, one or more different digital processing devices, one or more computing systems, one or more computer networks, and/or one or more communication networks.

In some embodiments, the digital processing device is communicatively coupled to a computer network ("network") by way of a communications interface suitable networks include Personal Area Networks (PANs), local area networks (L AN), Wide Area Networks (WANs), intranets, extranets, the internet (providing access to the world wide web), and combinations thereof.

Suitable digital processing devices include, by way of non-limiting example, server computers, desktop computers, laptop computers, notebook computers, mini notebook computers, netbook computers, nettablet computers, set-top box computers, media streaming devices, palmtop computers, internet appliances, mobile smartphones, tablet computers, and personal digital assistants in accordance with the description herein. Those skilled in the art will recognize that many smartphones are suitable for use in the system described herein. Those skilled in the art will also recognize that selected televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers include those known to those skilled in the art having a manual, tablet and convertible configuration.

In some embodiments, the digital processing device includes an operating system configured to execute executable instructions. An operating system is, for example, software including programs and data that manages the hardware of the device and provides services for the execution of application programs. Those skilled in the art will recognize that suitable server operating systems include, by way of non-limiting example, FreeBSD, OpenBSD,Linux、Mac OS XWindowsAndthose skilled in the art will recognize that suitable personal computer operating systems include, by way of non-limiting example Mac OSAnd UNIX-like operating systems, e.g.In some embodiments, the operating system is provided by cloud computing. Those skilled in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting example, a mobile smartphone operating systemOS、Research InBlackBerryWindowsOS、WindowsOS、Andthose skilled in the art will also recognize that suitable media streaming device operating systems include Apple, by way of non-limiting exampleGoogleGoogleAmazonAndin some cases, the operating system includes an internet of things (IoT) device. Non-limiting examples of IoT devices include AmazonMicrosoft's ofApple HomeAnd GoogleIn some cases, the devices, systems, and kits disclosed herein comprise virtual reality and/or augmented reality systems.

In some embodiments, the devices, systems, and kits disclosed herein comprise a storage and/or memory device. The storage and/or memory devices are one or more physical means for temporarily or permanently storing data or programs. In some embodiments, the device is a volatile memory and requires power to maintain the stored information. In some embodiments, the device is a non-volatile memory and retains stored information when the digital processing device is not powered. In a further embodiment, the non-volatile memory comprises flash memory. In some embodiments, the non-volatile memory comprises Dynamic Random Access Memory (DRAM). In some implementations, the non-volatile memory includes Ferroelectric Random Access Memory (FRAM). In some implementations, the non-volatile memory includes phase change random access memory (PRAM). In other embodiments, the device is a storage device, including, by way of non-limiting example, a CD-ROM, DVD, flash memory device, magnetic disk drive, magnetic tape drive, optical disk drive, and cloud-based storage. In further embodiments, the storage and/or memory devices are a combination of devices such as those disclosed herein.

In some embodiments, the display is a liquid crystal display (L CD). in further embodiments, the display is a thin film transistor liquid crystal display (TFT-L CD). in some embodiments, the display is an organic light emitting diode (O L ED) display.in various further embodiments, on the O L ED display is a passive matrix O L ED (PMO L ED) or active matrix O L ED (AMO L ED) display.

In some embodiments, the input device is a pointing device (pointing device), including a mouse, trackball, trackpad, joystick, game controller, or stylus, to name a non-limiting example.

Mobile application program

In some embodiments, the devices, systems, kits, and methods disclosed herein comprise or use a digital processing device, wherein the digital processing device is provided with executable instructions in the form of a mobile application. In some embodiments, the mobile application is provided to the mobile digital processing device at the time of manufacture. In other embodiments, the mobile application is provided to the mobile digital processing device over a computer network as described herein.

In view of the disclosure provided herein, mobile applications are created using hardware, languages, and development environments known in the art through techniques known to those skilled in the art. Those skilled in the art will recognize that mobile applications are written in several languages. By way of non-limiting example, suitable programming languages include C, C + +, C #, Objective-C, Java^TM、Javascript、Pascal、Object Pascal、Python^TMNet, WM L and XHTM L/HTM L with or without CSS, or combinations thereof.

Suitable mobile application development environments are available from several sources. By way of non-limiting example, commercially available development environments include AirplaySDK, alchemio, Gemini,other development environments are freely available, by way of non-limiting example, Celsius, Bedrop, Flash L ite,. NET Compact frame, Rhomobile, and word L light Mobile platformIncluding L azarus, mobilflex, MoSync, and phonegap. in addition, mobile device manufacturers distribute software developer toolkits including, by way of non-limiting example, iPhone and ipad (ios) SDK, Android^TMSDK、BlackSDK、BREW SDK、OS SDK, Symbian SDK, webOS SDK andMobile SDK。

those skilled in the art will recognize that several business forums may be used to distribute mobile applications, including by way of non-limiting exampleApp Store、Play、Chrome WebStore、BlackApp World, App Store for Palm devices, App Catalog for webOS, for mobile devicesMarkemplce forOvi Store and of the plantApps。

Referring to fig. 8A, in particular embodiments, mobile applications are configured to interface with, communicate with, and receive genetic and other information from the devices, systems, and kits disclosed herein. Fig. 8A is a diagram depicting various functions that a mobile application may optionally provide to a user. In this embodiment, the mobile application optionally provides: 1) personalized, customized user experience (UX) based on personal information and preferences of the user; 2) an interactive text, audio and/or video driven educational experience to inform the user how to use the device, system and kit; 3) a content platform that provides users with access to articles, news, media, games, etc.; and 4) tools for tracking and sharing information, test results, and events.

Referring to fig. 8B, in particular embodiments, the mobile application optionally includes an interactive interface that provides a step-by-step flow to guide the user in using the devices, systems, and kits disclosed herein. In various embodiments, the interactive flow includes text, images, animations, audio, video, etc. to inform and guide the user.

Referring to fig. 8C, in a particular embodiment, the mobile application optionally includes a home screen that allows a user to access the mobile application functionality disclosed herein. In this embodiment, the home screen includes personalized greetings and interface elements that allow the user to initiate tests, view current and historical test results, share test results, and interact with a larger community of users.

Referring to fig. 8D, in particular embodiments, the mobile application optionally includes a progress chart that informs the user of the status of the process for connecting to the device, system, or kit disclosed herein. In this embodiment, the figure shows all steps and indicates the current step. The steps are as follows: 1) pairing with a device through, for example, bluetooth; 2) detecting a sample in the device; and 3) waiting for the sample to be processed. In some embodiments, the graph is interactive, animated or enhanced with media or other content.

Referring to fig. 8E, in certain embodiments, the mobile application optionally includes a test report that is provided to the user to convey the test results. In this example, a report is provided to the user to let her know that she has a daughter. In some embodiments, the report is interactive, animated or augmented with media or other content, which may be personalized based on the test results.

Referring to FIG. 8F, in particular embodiments, the mobile application optionally includes a social sharing screen that allows users to access features to share test results, many services, platforms, and networks are suitable for sharing test results and other information and events, suitable social networks and sharing platforms include Facebook, YouTube, Twitter, L inkedIn, Pinterest, Google Plus +, Tumblr, Instagram, Reit, VK, Snapchat, Flickr, Vine, Meetup, ask.fm, Classmates, QQ, Wechat, Swarm of Foursquad, Kik, Yik Yak, Shots, Periscou, Medium, Soundcld, Tinder, WhatsA, Snap Chatt, Slack, music.

Referring to FIG. 8G, in certain embodiments, the mobile application optionally includes a home screen that allows the user to access additional functionality, such as a weblog and timeline of important information and events related to test results, which may optionally be shared. In various embodiments, suitable information and events include those related to clinical trial results, newly marketed therapies, nutrition, exercise, fetal development, health, and the like. In the case of pregnant subjects, information and events are organized into time line interfaces based on the time point (e.g., number of weeks) of pregnancy. In this embodiment, the home screen also includes access to user preferences and settings.

In some cases, the devices, systems, and kits disclosed herein are used according to the following methods.

II.Method of producing a composite material

In some aspects, the methods disclosed below employ the previously described devices, systems, and kits. Generally, the methods disclosed herein comprise obtaining a biological sample and detecting a component thereof. Obtaining a biological sample may occur in a clinical or laboratory setting. Alternatively, the acquisition may occur at a location remote from the clinical or laboratory environment, such as a home, school, farm, or battlefield, to name a non-limiting example. In some cases, the detection occurs in a clinical or laboratory setting. In other cases, detection occurs at a location remote from the clinical or laboratory environment. Other steps of the methods disclosed herein (e.g., amplifying nucleic acids) can occur in a clinical/laboratory setting or at a remote location.

In general, the methods disclosed herein include collecting and analyzing a relatively small volume of a biological sample. By way of non-limiting example, disclosed herein is a method comprising: obtaining a sample from a female subject in a non-laboratory environment, wherein the volume of the biological sample is no greater than about 300 μ Ι; amplifying at least one circulating cell-free nucleic acid in the sample to produce at least one amplification product; detecting the presence or absence of an amplification product comprising a sequence corresponding to the Y chromosome.

FIG. 2 illustrates a general flow diagram with various routes that may be followed by the methods, apparatus and systems disclosed herein. Initially, a sample is obtained in step 210. A minimum amount of sample must be obtained to gather useful information from the sample. The sample may be a biological sample as disclosed herein. The sample may be a raw, unprocessed sample (e.g., whole blood). The sample may be a processed sample (e.g., plasma). The amount of sample may be based on the type of sample. Generally, in step 220, a sample is processed or an analyte (e.g., a nucleic acid or other biomarker) is purified from the sample to produce an analyte that can be amplified and/or detected. Processing may include filtering the sample, binding components of the sample containing the analyte, binding the analyte, stabilizing the analyte, purifying the analyte, or a combination thereof. Non-limiting examples of sample components are cells, viral particles, bacterial particles, exosomes and nucleosomes. In some cases, the analyte is a nucleic acid, and it is amplified in step 240 to produce an amplicon for analysis. In other cases, the analyte may or may not be a nucleic acid, but is not amplified in any way. The analyte or amplicon is optionally modified (250) prior to detection and analysis in steps 260 and 270, respectively. In some cases, modification occurs during amplification (not shown). For example, the analyte or amplicon may be tagged or labeled. Detection may involve sequencing, target-specific probes, isothermal amplification and detection methods, quantitative PCR, or single molecule detection. Fig. 2 is provided as a broad overview of the apparatus and methods disclosed herein, but the apparatus and methods disclosed herein are not limited by fig. 2. The apparatus and method may contain additional components and steps, respectively, not shown in fig. 2.

Sample collection

In some cases, the methods disclosed herein comprise obtaining a biological sample described herein. Non-limiting examples of biological samples include blood, plasma, urine, saliva, vaginal fluid, interstitial fluid. In some cases, the methods disclosed herein comprise obtaining an environmental sample described herein. Non-limiting examples of environmental samples include wastewater, seawater, food, and beverages.

In some cases, the methods disclosed herein are performed at a single location, e.g., from obtaining to detecting. In some cases, the single location is a home. In some cases, a single site is not a medical, technical, or pathology laboratory. In some cases, the methods disclosed herein are performed entirely by a female subject. In some cases, the methods disclosed herein are performed by a subject who has not received any technical training to perform the methods. In some cases, the methods disclosed herein are performed by a subject who has not received any technical training to perform the methods, other than the set of instructions provided with the apparatus to perform the methods.

In some cases, the methods disclosed herein are performed with a device, system, or kit described herein. In some cases, the methods disclosed herein are performed remotely from a clinical environment, such as a medical clinic, hospital, scientific research laboratory, pathology laboratory, or clinical testing laboratory, by way of non-limiting example. In some cases, the methods disclosed herein are performed at home, school, or family planning center. In some cases, the method may be performed by a subject. In some cases, the methods disclosed herein are performed by a user who does not accept any technical training necessary to perform the method.

In general, the methods disclosed herein include obtaining, processing, and/or analyzing a relatively small volume of a biological sample. A small volume may also be referred to as a small input, low input, or low volume. In some cases, the methods disclosed herein comprise obtaining a volume of the biological sample, wherein the volume is less than 1 milliliter. In some cases, the methods disclosed herein are performed with no more than 1 milliliter of biological sample. In some cases, the methods disclosed herein are performed with no more than 100 μ Ι of biological sample. In some cases, the methods disclosed herein comprise obtaining a volume of the biological sample, wherein the volume falls within a range of sample volumes. In some cases, the sample volume ranges from about 1 μ l to about 1 ml. In some cases, the sample volume ranges from about 5 μ l to about 1 ml. In some cases, the sample volume ranges from about 1 μ l to about 900 μ l. In some cases, the sample volume ranges from about 1 μ l to about 800 μ l. In some cases, the sample volume ranges from about 1 μ l to about 700 μ l. In some cases, the sample volume ranges from about 1 μ l to about 600 μ l. In some cases, the sample volume ranges from about 1 μ l to about 500 μ l. In some cases, the sample volume ranges from about 1 μ l to about 400 μ l. In some cases, the sample volume ranges from about 1 μ l to about 300 μ l. In some cases, the sample volume ranges from about 1 μ l to about 200 μ l. In some cases, the sample volume ranges from about 1 μ l to about 150 μ l. In some cases, the sample volume ranges from 1 μ l to about 100 μ l. In some cases, the sample volume ranges from about 1 μ l to about 90 μ l. In some cases, the sample volume ranges from about 1 μ l to about 85 μ l. In some cases, the sample volume ranges from about 1 μ l to about 80 μ l. In some cases, the sample volume ranges from about 1 μ l to about 75 μ l. In some cases, the sample volume ranges from about 1 μ l to about 70 μ l. In some cases, the sample volume ranges from about 1 μ l to about 65 μ l. In some cases, the sample volume ranges from about 1 μ l to about 60 μ l. In some cases, the sample volume ranges from about 1 μ l to about 55 μ l. In some cases, the sample volume ranges from about 1 μ l to about 50 μ l. In some cases, the sample volume ranges from about 5 μ l to about 45 μ l. In some cases, the sample volume ranges from about 5 μ l to about 40 μ l. In some cases, the sample volume ranges from about 15 μ l to about 150 μ l. In some cases, the sample volume ranges from 15 μ l to about 100 μ l. In some cases, the sample volume ranges from about 15 μ l to about 90 μ l. In some cases, the sample volume ranges from about 15 μ l to about 85 μ l. In some cases, the sample volume ranges from about 15 μ l to about 80 μ l. In some cases, the sample volume ranges from about 15 μ l to about 75 μ l. In some cases, the sample volume ranges from about 15 μ l to about 70 μ l. In some cases, the sample volume ranges from about 15 μ l to about 65 μ l. In some cases, the sample volume ranges from about 15 μ l to about 60 μ l. In some cases, the sample volume ranges from about 15 μ l to about 55 μ l. In some cases, the sample volume ranges from about 15 μ l to about 50 μ l. In some cases, the sample volume ranges from about 10 μ l to about 45 μ l. In some cases, the sample volume ranges from about 10 μ l to about 40 μ l.

In some aspects, the methods described herein include obtaining a fluid sample from a subject with a handheld device, wherein the fluid sample has a volume of no greater than about 300 μ L, sequencing at least one cell-free nucleic acid in the fluid sample with the handheld device, detecting the presence or absence of a sequence corresponding to a sequence of interest via a display in the handheld device to determine genetic information about the subject, and transferring the genetic information to another subject with the handheld device.

In some cases, the methods disclosed herein comprise obtaining a blood sample. In some cases, obtaining blood does not include a phlebotomy. In some cases, the subject is obtained by pressing his skin against the transcutaneous penetration device of the handheld device. Typically, the transcutaneous puncture device comprises at least one of a needle, a microneedle or an array of needles. In some cases, the subject presses a finger, toe, arm, shoulder, or palm against the transcutaneous device. In some cases, the subject presses a finger against the transcutaneous puncture device. In some cases, the subject presses their skin against the transcutaneous puncture device no more than once. In some cases, the subject presses their skin against the transcutaneous puncture device no more than twice. In some cases, the methods include obtaining a blood sample and sending the blood sample or a component thereof (e.g., plasma/serum) to a location remote from the site of the obtaining step (e.g., a laboratory, clinic, or research center) for additional processing and analysis. In other cases, the method includes detecting the test result at the location of the obtaining step using, for example, an apparatus disclosed herein.

In some cases, the methods disclosed herein comprise obtaining a blood sample by finger prick. In some cases, the methods disclosed herein comprise obtaining a blood sample by multiple finger sticks. In some cases, the methods disclosed herein comprise obtaining a blood sample from no more than 2 finger sticks. In some cases, the methods disclosed herein comprise obtaining a blood sample from no more than 3 finger sticks. In some cases, the methods disclosed herein comprise obtaining a blood sample by a single finger prick. In some cases, the methods disclosed herein comprise obtaining a blood sample by no more than a single finger prick. In some cases, the methods disclosed herein include obtaining capillary blood (e.g., blood obtained from a finger). In some cases, the method includes squeezing or expressing blood from the needle to obtain a desired blood volume. Although finger prick is a common method of obtaining capillary blood, other parts of the body will also be suitable, e.g. toes, palm, heel, arm, shoulder. In some cases, the methods disclosed herein comprise obtaining a blood sample without venotomy. In some cases, the methods disclosed herein do not include obtaining venous blood (e.g., blood obtained from a vein).

In some cases, the methods disclosed herein include obtaining at least about 1 μ L blood to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 5 μ L blood to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 15 μ L blood to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 15 μ L blood to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 20 μ L blood to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 20 μ L blood to provide test results with at least about 95% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 20 μ 464 blood to provide test results with at least about 100 μ 465% confidence or accuracy in some cases, the devices, systems, and kits require at least about 20 μ 464 to provide test results with at least about 100 μ 4695% confidence or accuracy in some cases, the devices, about 20 μ 4695% confidence or about 100 μ 464 to provide test results with at least about 100 μ 4695% confidence in some cases, the test results with at least about 100 μ 465 to provide test results in some cases.

In some cases, the methods disclosed herein comprise isolating cells from a biological sample. In some cases, the methods disclosed herein comprise separating a portion of the cell-free sample from a portion of the cell-containing sample. The method may comprise treating the cells and/or analysing the contents of the cells. The processing or analysis may occur within the devices or systems disclosed herein. In some cases, the cells are retained or preserved for subsequent analysis outside of the device or system.

In some cases, the methods disclosed herein comprise obtaining plasma that comprises about 55% of whole blood in some cases, the devices, systems, kits, and methods disclosed herein require at least about 3 μ L plasma to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 8 μ L plasma to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 8 μ L0 plasma to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 12 μ L1 plasma to provide test results with at least about 90% confidence or accuracy in some cases, the devices, systems, and kits disclosed herein require at least about 12 μ L to provide test results with at least about 95% confidence or accuracy in some cases, the devices, systems, and kits require at least about 12 μ 465% confidence to provide test results with at least about 99 μ 4660 to provide test results with at least about 95% confidence or accuracy in some cases, about 90% to provide test results with at least about 90 μ 4695% accuracy in some cases, about 95% confidence or accuracy in some cases, about 99 μ 465% to provide test results.

In some cases, the biological sample evaluated using the methods, devices, systems, and kits disclosed herein is urine, and the volume of urine used is about 0.25 μ Ι to 1 milliliter. In some cases, the volume of urine used is from about 0.25 μ l to about 1 ml. In some cases, the volume of urine used is at least about 0.25 μ l. In some cases, the volume of urine used is at most about 1 milliliter. In some cases, the urine used has a volume of about 0.25 μ l to about 0.5 μ l, about 0.25 μ l to about 0.75 μ l, about 0.25 μ l to about 1 μ l, about 0.25 μ l to about 5 μ l, about 0.25 μ l to about 10 μ l, about 0.25 μ l to about 50 μ l, about 0.25 μ l to about 100 μ l, about 0.25 μ l to about 150 μ l, about 0.25 μ l to about 200 μ l, about 0.25 μ l to about 500 μ l, about 0.25 μ l to about 1ml, about 0.5 μ l to about 0.75 μ l, about 0.5 μ l to about 1 μ l, about 0.5 μ l to about 5 μ l, about 0.5 μ l to about 10 μ l, about 0.5 μ l to about 50 μ l, about 0.5 μ l to about 100 μ l, about 0.5 μ l to about 0.5 μ l, about 0.5 μ l to about 5 μ l, about 0.75 μ l to about 0.75 μ l, about 0.5 μ l to about 0 μ l, about 0.5 μ l to about 5 μ l, about 0.75 μ l to about 0.75 μ l, about 0.5 μ l to about 0.1 μ l, about 0.5 μ l to about 0.75 μ l, about 0.1 μ l to about 0.1 μ l, about 0.75. mu.l to about 100. mu.l, about 0.75. mu.l to about 150. mu.l, about 0.75. mu.l to about 200. mu.l, about 0.75. mu.l to about 500. mu.l, about 0.75. mu.l to about 1ml, about 1. mu.l to about 5. mu.l, about 1. mu.l to about 10. mu.l, about 1. mu.l to about 50. mu.l, about 1. mu.l to about 100. mu.l, about 1. mu.l to about 150. mu.l, about 1. mu.l to about 200. mu.l, about 1. mu.l to about 500. mu.l, about 1. mu.l to about 1ml, about 5. mu.l to about 10. mu.l, about 5. mu.l to about 50. mu.l, about 5. mu.l to about 100. mu.l, about 5. mu.l to about 150. mu.l, about 5. mu.l to about 200. mu.l, about 5. mu.l to about 500. mu.l, about 5. mu.l to about 1. mu.l, about 10. mu.l to about 10. mu.l, about 10. mu., About 50. mu.l to about 100. mu.l, about 50. mu.l to about 150. mu.l, about 50. mu.l to about 200. mu.l, about 50. mu.l to about 500. mu.l, about 50. mu.l to about 1ml, about 100. mu.l to about 150. mu.l, about 100. mu.l to about 200. mu.l, about 100. mu.l to about 500. mu.l, about 100. mu.l to about 1ml, about 150. mu.l to about 200. mu.l, about 150. mu.l to about 500. mu.l, about 150. mu.l to about 1ml, about 200. mu.l to about 500. mu.l, about 200. mu.l to about 1ml, or about 500. mu.l to about 1 ml. In some cases, the volume of urine used is about 0.25. mu.l, about 0.5. mu.l, about 0.75. mu.l, about 1. mu.l, about 5. mu.l, about 10. mu.l, about 50. mu.l, about 100. mu.l, about 150. mu.l, about 200. mu.l, about 500. mu.l, or about 1 ml.

In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains an amount of cell-free nucleic acid. In some cases, the cell-free nucleic acid comprises DNA. In some cases, the cell-free nucleic acid comprises RNA. In some cases, cell-free nucleic acids include DNA and RNA. In some cases, the cell-free nucleic acid comprises cell-free fetal nucleic acid. In some cases, the amount of cell-free nucleic acid falls within a certain range. In some cases, the range is about 1pg to about 10 pg. In some cases, the range is about 1pg to about 50 pg. In some cases, the range is about 1pg to about 100 pg. In some cases, the range is about 1pg to about 1 ng. In some cases, the range is about 2pg to about 10 pg. In some cases, the range is about 1pg to about 1 ng. In some cases, the range is about 2pg to about 100 pg. In some cases, the range is about 3pg to about 10 pg. In some cases, the range is about 3pg to about 30 pg. In some cases, the range is about 3pg to about 100 pg. In some cases, the range is about 3pg to about 300 pg. In some cases, the range is about 3pg to about 1 ng. In some cases, the range is about 3pg to about 2 ng. In some cases, the range is about 3pg to about 3 ng. In some cases, the range is about 3pg to about 4 ng. In some cases, the range is about 3pg to about 5 ng. In some cases, the range is about 3pg to about 10 ng. In some cases, the method comprises obtaining less than about 10ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining less than about 7ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining less than about 5ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining less than about 1ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining no more than about 10ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining no more than about 7ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining no more than about 5ng of cell-free fetal nucleic acid. In some cases, the method comprises obtaining no more than about 1ng of cell-free fetal nucleic acid.

In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains at least one cell-free fetal nucleic acid comprising a sequence unique to the Y chromosome. In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains from about 1 to about 5 cell-free fetal nucleic acids comprising a sequence unique to the Y chromosome. In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains from about 1 to about 15 cell-free fetal nucleic acids comprising a sequence unique to the Y chromosome. In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains from about 1 to about 25 cell-free fetal nucleic acids comprising a sequence unique to the Y chromosome. In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains from about 1 to about 100 cell-free fetal nucleic acids comprising a sequence unique to the Y chromosome. In some cases, the methods disclosed herein comprise obtaining a biological sample from a female subject, wherein the biological sample contains from about 5 to about 100 cell-free fetal nucleic acids comprising a sequence unique to the Y chromosome.

By way of non-limiting example, a method may include obtaining a fluid sample from a female pregnant subject using a handheld device, wherein the fluid sample has a volume of no greater than about 300 μ L, sequencing at least one cell-free nucleic acid in the fluid sample using the handheld device, detecting the presence or absence of a sequence corresponding to the Y chromosome by a display in the handheld device, thereby determining the gender of a fetus within the female pregnant subject, and transmitting the gender to another subject via the handheld device.

Also by way of non-limiting example, a method may comprise obtaining a biological sample from a female subject, wherein the volume of the biological sample is no greater than about 120 μ Ι; contacting the sample with an oligonucleotide primer comprising a sequence corresponding to the Y chromosome to amplify at least one circulating cell-free nucleic acid in the sample; detecting the absence of the amplification product, thereby indicating that the fetus is female. The obtaining, contacting and detecting may be performed with a single device.

Isolation and purification of nucleic acids and other biomarkers

In some cases, the methods disclosed herein comprise isolating or purifying nucleic acids from one or more non-nucleic acid components of a biological sample. Non-nucleic acid components may also be considered as undesirable substances. Non-limiting examples of non-nucleic acid components include cells (e.g., blood cells), cell fragments, extracellular vesicles, lipids, proteins, or combinations thereof. Additional non-nucleic acid components are described herein and throughout. It should be noted that although the methods may include isolating/purifying nucleic acids, they may also include analyzing non-nucleic acid components of the sample for substances that are deemed undesirable in the nucleic acid purification step. Isolation or purification may include removal of components of the biological sample that would inhibit, interfere with, or otherwise be detrimental to subsequent processing steps, such as nucleic acid amplification or detection.

The isolation or purification can be performed using the apparatus or system disclosed herein. The isolation or purification can be performed within an apparatus or system disclosed herein. The separation and/or purification can be performed by using the sample purifiers disclosed herein. In some cases, isolating or purifying nucleic acids comprises removing non-nucleic acid components from a biological sample described herein. In some cases, isolating or purifying nucleic acids includes discarding non-nucleic acid components from the biological sample. In some cases, the isolating or purifying includes collecting, processing, and analyzing non-nucleic acid components. In some cases, non-nucleic acid components may be considered biomarkers as they provide additional information about the subject.

In some cases, isolating or purifying the nucleic acid comprises lysing the cells. In some cases, isolating or purifying nucleic acids avoids cell lysis. In some cases, isolating or purifying nucleic acid does not include lysing the cells. In some cases, isolating or purifying nucleic acids does not include an active step intended to lyse the cells. In some cases, isolating or purifying nucleic acid does not include intentionally lysing the cells. Intentionally lysing cells may include mechanically disrupting the cell membrane (e.g., shearing). Intentionally lysing cells may include contacting the cells with a lysis reagent. Exemplary lysis reagents are described herein.

In some cases, isolating or purifying nucleic acids involves cleaving in solution and performing sequence-specific capture of the target nucleic acid with a "bait" that is then bound to a solid support such as a magnetic bead, e.g., L egler et al, scientific bead-based capture of free genomic DNA from a mechanical plant, Transfusion and Apheresis Science 40(2009),

153-157. In some cases, the method comprises performing sequence-specific capture in the presence of a recombinase or helicase. The use of a recombinase or helicase can avoid the need for thermal denaturation of the nucleic acid and speed up the detection step.

In some cases, isolating or purifying comprises isolating a component of a biological sample disclosed herein. By way of non-limiting example, the separating or purifying may include separating plasma from blood. In some cases, isolating or purifying comprises centrifuging the biological sample. In some cases, isolating or purifying includes filtering the biological sample to isolate components of the biological sample. In some cases, the isolating or purifying comprises filtering the biological sample to remove non-nucleic acid components from the biological sample. In some cases, isolating or purifying includes filtering the biological sample to capture nucleic acids from the biological sample.

In some cases, the biological sample is blood, and isolating or purifying the nucleic acid comprises obtaining or isolating plasma from the blood. Obtaining plasma may include separating plasma from cellular components of the blood sample. Obtaining plasma may include centrifuging the blood, filtering the blood, or a combination thereof. Obtaining plasma may include subjecting the blood to gravity (e.g., sedimentation). Obtaining plasma may include subjecting the blood to a material that wicks away a portion of the blood from non-nucleic acid components of the blood. In some cases, the method comprises subjecting the blood to vertical filtration. In some cases, a method includes subjecting blood to a sample purifier comprising a filtration matrix for receiving whole blood, the filtration matrix having a pore size that prevents passage of cells, while plasma can pass through the filtration matrix uninhibited. Such vertical filtration and filtration matrices are described for the devices disclosed herein.

In some cases, isolating or purifying comprises subjecting the biological sample or a portion thereof or a modified form thereof to a binding moiety. The binding moiety may be capable of binding to a component of the biological sample and removing it to produce a modified sample that has removed cells, cell fragments, nucleic acids, or proteins that are not desired or of interest. In some cases, isolating or purifying includes subjecting the biological sample to a binding moiety to reduce unwanted substances or non-nucleic acid components in the biological sample. In some cases, isolating or purifying comprises subjecting the biological sample to a binding moiety to produce a modified sample enriched for the target cell, target cell fragment, target nucleic acid, or target protein. By way of non-limiting example, isolating or purifying may include subjecting the biological sample to a binding moiety for capturing placental-cultured platelets, which may contain fetal DNA or RNA fragments. The resulting cell-bound binding moieties can be captured/enriched with antibodies or other methods, e.g., low speed centrifugation.

The isolating or purifying may include capturing extracellular vesicles or extracellular microparticles in the biological sample with the binding moiety. In some cases, the extracellular vesicles contain at least one of DNA and RNA. In some cases, the extracellular vesicles are derived from a fetus/placenta. The method may include capturing extracellular vesicles or extracellular microparticles in a biological sample from a maternal cell. In some cases, the methods disclosed herein comprise capturing and discarding extracellular vesicles or extracellular microparticles from maternal cells to enrich the fetal/placental nucleic acids of the sample.

In some cases, the method comprises capturing nucleosomes in the biological sample and analyzing nucleic acids attached to the nucleosomes. In some cases, the method comprises capturing exosomes in the biological sample and analyzing nucleic acids attached to the exosomes. Capturing nucleosomes and/or exosomes may eliminate the need for a lysis step or reagent, thereby simplifying the method and reducing the time from sample collection to detection.

In some cases, the method includes subjecting the biological sample to a cell binding moiety to capture placental-cultured platelets, which may contain fetal DNA or RNA fragments. Capturing can include contacting placenta-cultured platelets with a binding moiety (e.g., an antibody to a cell surface marker), subjecting the biological sample to low speed centrifugation, or a combination thereof. In some cases, the binding moiety is attached to a solid support disclosed herein, and the method comprises separating the solid support from the remainder of the biological sample after the binding moiety is contacted with the biological sample.

In some cases, the isolating or purifying includes reducing unwanted non-nucleic acid components from the biological sample. In some cases, the isolation or purification includes removing unwanted non-nucleic acid components from the biological sample. In some cases, isolating or purifying comprises removing at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the undesired non-nucleic acid components from the biological sample. In some cases, the isolating or purifying comprises removing at least 95% of the undesired non-nucleic acid components from the biological sample. In some cases, the isolating or purifying comprises removing at least 97% of the undesired non-nucleic acid components from the biological sample. In some cases, the isolating or purifying comprises removing at least 98% of the undesired non-nucleic acid components from the biological sample. In some cases, the isolating or purifying comprises removing at least 99% of the undesired non-nucleic acid components from the biological sample.

In some cases, the methods disclosed herein comprise purifying nucleic acids in a sample. In some cases, purification includes washing the nucleic acid with a wash buffer. In some cases, purification does not include washing the nucleic acid with a wash buffer. In some embodiments, purifying comprises capturing nucleic acids with a nucleic acid capture moiety to produce captured nucleic acids. Non-limiting examples of nucleic acid capture moieties are silica particles and paramagnetic particles. In some embodiments, purification includes passing the sample containing the captured nucleic acids through a hydrophobic phase (e.g., a liquid or wax). The hydrophobic phase retains impurities in the sample that would otherwise inhibit further manipulation of the nucleic acid (e.g., amplification, sequencing).

In some cases, the methods disclosed herein comprise removing a nucleic acid component from a biological sample described herein. In some cases, the removed nucleic acid component is discarded. By way of non-limiting example, a method may comprise analyzing only DNA. Thus, RNA is not required and produces undesirable background noise or contamination of DNA. In some cases, the methods disclosed herein comprise removing RNA from a biological sample. In some cases, the methods disclosed herein comprise removing mRNA from a biological sample. In some cases, the methods disclosed herein comprise removing micrornas from a biological sample. In some cases, the methods disclosed herein comprise removing maternal RNA from the biological sample. In some cases, the methods disclosed herein comprise removing DNA from a biological sample. In some cases, the methods disclosed herein comprise removing maternal DNA from a biological sample of a pregnant subject. In some cases, removing the nucleic acid component comprises contacting the nucleic acid component with an oligonucleotide capable of hybridizing to the nucleic acid, wherein the oligonucleotide is conjugated, attached, or bound to a capture device (e.g., a bead, column, matrix, nanoparticle, magnetic particle, etc.).

In some cases, removing the nucleic acid component includes separating the nucleic acid component on the gel by size. For example, the circulating cell-free fetal DNA fragments are smaller than the circulating maternal DNA fragments. The length of the circulating cell-free fetal DNA fragments is typically less than 200 base pairs. In some cases, the methods disclosed herein comprise removing cell-free DNA from the biological sample, wherein the cell-free DNA has a minimum length. In some cases, the minimum length is about 50 base pairs. In some cases, the minimum length is about 100 base pairs. In some cases, the minimum length is about 110 base pairs. In some cases, the minimum length is about 120 base pairs. In some cases, the minimum length is about 140 base pairs. In some cases, the methods disclosed herein comprise selecting cell-free DNA from a biological sample, wherein the cell-free DNA has a maximum length. In some cases, the maximum length is about 180 base pairs. In some cases, the maximum length is about 200 base pairs. In some cases, the maximum length is about 220 base pairs. In some cases, the maximum length is about 240 base pairs. In some cases, the maximum length is about 300 base pairs. In some cases, the maximum length is about 400 base pairs. In some cases, the maximum length is about 500 base pairs. Size-based separation will be useful for other classes of nucleic acids with a limited size range, which are well known in the art (e.g., micrornas).

Amplification of nucleic acids

In some cases, the methods disclosed herein comprise amplifying at least one nucleic acid in a sample to produce at least one amplification product. The at least one nucleic acid may be a cell-free nucleic acid. The sample may be a biological sample as disclosed herein or a fraction or portion thereof. The sample may be an environmental sample. In some cases, a method includes generating a copy of a nucleic acid in a sample and amplifying the copy to generate at least one amplification product. In some cases, a method includes generating a reverse transcript of a nucleic acid in a sample and amplifying the reverse transcript to generate at least one amplification product.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of interest. In some cases, the methods disclosed herein comprise quantifying a circulating cell-free nucleic acid comprising a sequence of interest. In some cases, the methods disclosed herein comprise amplifying a circulating cell-free nucleic acid comprising a sequence of interest. In some cases, amplification comprises polymerase-mediated amplification with primers that anneal to the sense and antisense strands corresponding to the sequence of interest. In some cases, detecting or quantifying comprises hybridizing a circulating cell-free nucleic acid comprising a sequence of interest to an oligonucleotide probe. The oligonucleotide probe can anneal to at least a portion of the sequence of interest or its complement. By way of non-limiting example, the sequence of interest can be a sequence of a repeat region (e.g., multiple copies of the sequence of interest) or a Y chromosome-specific sequence.

In some cases, the methods disclosed herein comprise amplifying the nucleic acid at least at one temperature. In some cases, the methods disclosed herein comprise amplifying a nucleic acid at a single temperature (e.g., isothermal amplification). In some cases, the methods disclosed herein comprise amplifying the nucleic acid, wherein the amplification occurs at no more than two temperatures. Amplification may occur in one step or in multiple steps. Non-limiting examples of amplification steps include double strand denaturation, primer hybridization, and primer extension.

In some cases, at least one step of amplification occurs at room temperature. In some cases, all steps of amplification occur at room temperature. In some cases, at least one step of amplification occurs within a temperature range. In some cases, all steps of amplification occur within a temperature range. In some cases, the temperature ranges from about 0 ℃ to about 100 ℃. In some cases, the temperature ranges from about 15 ℃ to about 100 ℃. In some cases, the temperature ranges from about 25 ℃ to about 100 ℃. In some cases, the temperature ranges from about 35 ℃ to about 100 ℃. In some cases, the temperature ranges from about 55 ℃ to about 100 ℃. In some cases, the temperature range is from about 65 ℃ to about 100 ℃. In some cases, the temperature ranges from about 15 ℃ to about 80 ℃. In some cases, the temperature range is from about 25 ℃ to about 80 ℃. In some cases, the temperature ranges from about 35 ℃ to about 80 ℃. In some cases, the temperature range is from about 55 ℃ to about 80 ℃. In some cases, the temperature range is from about 65 ℃ to about 80 ℃. In some cases, the temperature range is from about 15 ℃ to about 60 ℃. In some cases, the temperature range is from about 25 ℃ to about 60 ℃. In some cases, the temperature range is from about 35 ℃ to about 60 ℃. In some cases, the temperature range is from about 15 ℃ to about 40 ℃. In some cases, the temperature ranges from about-20 ℃ to about 100 ℃. In some cases, the temperature ranges from about-20 ℃ to about 90 ℃. In some cases, the temperature ranges from about-20 ℃ to about 50 ℃. In some cases, the temperature ranges from about-20 ℃ to about 40 ℃. In some cases, the temperature ranges from about-20 ℃ to about 10 ℃. In some cases, the temperature ranges from about 0 ℃ to about 100 ℃. In some cases, the temperature range is from about 0 ℃ to about 40 ℃. In some cases, the temperature ranges from about 0 ℃ to about 30 ℃. In some cases, the temperature ranges from about 0 ℃ to about 20 ℃. In some cases, the temperature range is from about 0 ℃ to about 10 ℃. In some cases, the temperature ranges from about 15 ℃ to about 100 ℃. In some cases, the temperature ranges from about 15 ℃ to about 90 ℃. In some cases, the temperature ranges from about 15 ℃ to about 80 ℃. In some cases, the temperature ranges from about 15 ℃ to about 70 ℃. In some cases, the temperature range is from about 15 ℃ to about 60 ℃. In some cases, the temperature ranges from about 15 ℃ to about 50 ℃. In some cases, the temperature ranges from about 15 ℃ to about 30 ℃. In some cases, the temperature range is from about 10 ℃ to about 30 ℃. In some cases, the methods disclosed herein are performed at room temperature, without the need for cooling, freezing, or heating.

In some cases, amplifying the nucleic acid comprises contacting the nucleic acid with random oligonucleotide primers. Amplification with multiple random primers typically results in non-targeted amplification of multiple nucleic acids of different sequences or overall amplification of most nucleic acids in a sample. In some cases, amplification comprises contacting a cell-free nucleic acid molecule disclosed herein with random oligonucleotide primers. In some cases, amplifying comprises contacting the cell-free fetal nucleic acid molecules disclosed herein with random oligonucleotide primers. In some cases, amplification comprises contacting a labeled nucleic acid molecule disclosed herein with a random oligonucleotide primer.

In some cases, amplification comprises targeted amplification (e.g., selector methods (described in US 6558928), molecular inversion probes). In some cases, amplifying the nucleic acid comprises contacting the nucleic acid with at least one primer having a sequence corresponding to the target chromosomal sequence. Exemplary chromosomal sequences are disclosed herein. In some cases, amplifying comprises contacting the nucleic acid with at least one primer having a sequence corresponding to a non-target chromosomal sequence. In some cases, amplification comprises contacting the nucleic acid with no more than one pair of primers, wherein each primer of the pair of primers comprises a sequence corresponding to a sequence on a target chromosome disclosed herein. In some cases, amplifying comprises contacting the nucleic acids with a plurality of sets of primers, wherein each of the first pairs in the first set and each of the pairs in the second set are different.

In some cases, amplification includes multiplexing (nucleic acid amplification of multiple nucleic acids in one reaction). In some cases, multiplexing comprises contacting nucleic acids of the biological sample with a plurality of oligonucleotide primer pairs. In some cases, multiplexing comprises contacting a first nucleic acid and a second nucleic acid, wherein the first nucleic acid corresponds to a first sequence and the second nucleic acid corresponds to a second sequence. In some cases, the first sequence and the second sequence are the same. In some cases, the first sequence and the second sequence are different. In some cases, amplification does not include multiplexing. In some cases, amplification does not require multiplexing. In some cases, the amplification comprises nested primer amplification.

In some cases, the method comprises amplifying nucleic acids in the sample, wherein amplifying comprises contacting the sample with at least one oligonucleotide primer, wherein the at least one oligonucleotide primer is not active or extendable until contacted with the sample. In some cases, amplification comprises contacting the sample with at least one oligonucleotide primer, wherein the at least one oligonucleotide primer is not active or extendable until exposed to a selected temperature. In some cases, amplification comprises contacting the sample with at least one oligonucleotide primer, wherein the at least one oligonucleotide primer is not active or extendable until contacted with an activating reagent. By way of non-limiting example, at least one oligonucleotide primer may comprise a blocking group. The use of such oligonucleotide primers may minimize primer dimer, allow identification of unused primers, and/or avoid erroneous results caused by unused primers. In some cases, amplification comprises contacting the sample with at least one oligonucleotide primer comprising a sequence corresponding to a sequence on a target chromosome disclosed herein.

In some cases, the tag comprises a nucleic acid having a universal sequence that does not correspond to any particular target sequence.

In some cases, the oligonucleotide primer comprises an oligonucleotide tag having a sequence that is not specific for a sequence on the Y chromosome. Such tags may be referred to as generic tags. In other cases, where the target sequence or sequence of interest corresponds to a chromosome other than the Y chromosome, the tag may be specific for a sequence on the Y chromosome. In some cases, the tag is specific for a sequence other than the sequence of interest, but corresponds to the same chromosome as the sequence of interest. In some cases, the tag is not specific for a sequence on a human chromosome. Alternatively or additionally, the oligonucleotide primer comprises an oligonucleotide tag having a sequence specific for a sequence on the Y chromosome. In some cases, the method comprises contacting the sample with a tag and at least one oligonucleotide primer comprising a sequence corresponding to a sequence on the Y chromosome, wherein the tag is separated from the oligonucleotide primer. In some cases, the tag is incorporated into the amplification product produced by extension of the oligonucleotide primer after the oligonucleotide primer hybridizes to the Y chromosome fragment.

In some cases, amplifying comprises contacting the sample with at least one primer having a sequence corresponding to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with at least one primer having a sequence complementary to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with at least one primer having a sequence identical to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with at least one primer having a sequence at least 90% identical to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with at least one primer having a sequence at least 75% identical to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with at least one primer having a sequence at least 60% identical to a sequence on the Y chromosome. In some cases, amplifying comprises contacting the sample with no more than one pair of primers, wherein each primer of the pair of primers comprises a sequence corresponding to a sequence on the Y chromosome.

In some cases, the methods disclosed herein include the use of multiple tags, thereby increasing at least one of the accuracy of the methods, the speed of the methods, and the information obtained by the methods. In some cases, the methods disclosed herein include the use of multiple tags, thereby reducing the sample volume required to obtain reliable results. In some cases, the plurality of tags includes at least one capture tag. In some cases, the plurality of tags includes at least one detection tag. Capture tags are typically used to separate or isolate a particular sequence or region from other regions. A typical example of a capture tag is biotin (biotin can be captured using, for example, a streptavidin-coated surface). Examples of detection tags are digoxigenin and fluorescent tags. The detection label can be detected directly (e.g., laser irradiation and/or measurement of emitted light) or indirectly by an antibody that carries or interacts with a secondary detection system such as a luminescence assay or an enzymatic assay. In some cases, the plurality of labels includes a combination of at least one capture label (a label for separating the analyte) and at least one detection label (a label for detecting the analyte). In some cases, a single tag acts as both a detection tag and a capture tag.

In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag comprises a first oligonucleotide that is complementary to a sense strand of the circulating cell-free nucleic acid and the second capture tag comprises a second oligonucleotide that is complementary to an antisense strand of the circulating cell-free nucleic acid. In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag carries the same label as the second tag. In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag carries a different label than the second tag. In some cases, the tags are the same and there is a single qualitative or quantitative signal that is a collection of all probes/regions detected. In some cases, the labels are different. One tag may be used for purification and one tag may be used for detection. In some cases, the first oligonucleotide tag is specific for a region (e.g., cfDNA fragment) and carries a fluorescent label, while the second oligonucleotide tag is specific for an adjacent region and carries the same fluorescent label, as only a collective signal is required. In other cases, the first oligonucleotide tag is specific for a region (e.g., cfDNA fragment) and carries a fluorescent label, while the second oligonucleotide tag is specific for an adjacent region and carries a different fluorescent label to detect the two different regions.

A number of isothermal amplification Methods are known in the art, each having different considerations and offering different advantages and discussed in the literature, e.g., Zanoli and Spoto,2013, "isothermal amplification Methods for the Detection of Nucleic Acids in microfluidic devices," Biosensors 3:18-43, and Fakrudtin, et al, 2013, "Alternative Methods of Polymerase Chain Reaction (PCR)," Journal of Nucleic and biological Sciences 5 (4: AMP 245, each of which is incorporated herein by reference in its entirety, isothermal amplification Methods for Reaction (NASA); amplification Methods for isothermal amplification of Nucleic Acids in PCR), and amplification Methods for isothermal amplification of Nucleic Acids in PCR (PCR) using isothermal displacement amplification of loop-mediated amplification sequences (PNA) L, and amplification Methods for amplification of Nucleic Acids in PCR (PCR) using isothermal displacement amplification of amplified sequences (PCR) in some cases, including amplification of amplified PCR-amplified Nucleic Acids in PCR) using isothermal displacement amplification (PCR) polymerase Chain displacement amplification Methods (PCR) in which the PCR amplification Methods are incorporated herein in their entirety by recombinase polymerase Chain displacement amplification (PCR) polymerase Chain displacement amplification Methods (PCR) and amplification Methods (PCR) in which are selected from the group consisting of isothermal amplification of amplified PCR) including the PCR amplification of Nucleic Acids in PCR amplification of PCR (PCR) using a polymerase Chain displacement amplification of a polymerase Chain amplification target (PCR) and amplification method (PCR) for amplification of Nucleic Acids (PCR) using a polymerase Chain displacement amplification method (PCR) for amplification of Nucleic Acids in which is known in the amplification of Nucleic Acids.

In some cases, the Amplification method used is L AMP (see, e.g., Notomi, et al, 2000, "L oop multiplexed Amplification" NAR 28(12): e63I-vii, and U.S. patent No. 6,410,278, "Process for synthesizing nucleic acid" (each incorporated herein by reference in its entirety.) L AMP is a one-step Amplification system using automated cycle strand displacement deoxyribonucleic acid (DNA) synthesis, L AMP in some cases is performed at 60-65 ℃ for 45-60min in the presence of a thermostable polymerase such as bacillus stearothermophilus (Bst) DNA polymerase I, deoxyribo triphosphate (dNTP), specific primers, and a target DNA template, in some cases the template is, and the reverse transcriptase activity and the polymerase activity are used, e.g., Bca polymerase, and the reverse transcriptase polymerase activity is used for the step of reverse transcriptase-DNA synthesis or the reverse transcriptase-displacing polymerase is used for the step of RNA-DNA synthesis.

In some cases, the amplification reaction is performed using L AMP at about 55 ℃ to about 70 ℃, in some cases, the L AMP reaction is performed at a temperature of 55 ℃ or higher, in some cases, the L AMP reaction is performed at a temperature of 70 ℃ or lower, in some cases, the L AMP reaction is performed at about 55 ℃ to about 57 ℃, about 55 ℃ to about 59 ℃, about 55 ℃ to about 60 ℃, about 55 ℃ to about 61 ℃, about 55 ℃ to about 62 ℃, about 55 ℃ to about 63 ℃, about 55 ℃ to about 64 ℃, about 55 ℃ to about 65 ℃, about 55 ℃ to about 66 ℃, about 55 ℃ to about 68 ℃, about 55 ℃ to about 70 ℃, about 57 ℃ to about 59 ℃, about 57 ℃ to about 62 ℃, about 57 ℃ to about 63 ℃, about 57 ℃ to about 64 ℃, about 57 ℃ to about 65 ℃, about 57 ℃ to about 66 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 61 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 62 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 61 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 61 ℃, about 60 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 60 ℃, about 61 ℃, about 60 ℃, about 61 ℃ to about 61 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 59 ℃ to about 61 ℃, about 60 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 60 ℃, about 61 ℃ to about 60 ℃, about 59 ℃ to about 60 ℃, about 61 ℃ to about 60 ℃, about 60 ℃, about 61 ℃ to about 60 ℃, about 59 ℃ to about 60 ℃, about 61 ℃ to about 61 ℃, about 61 ℃ to about, about 61 ℃ to.

In some cases, the amplification reaction is performed using L AMP for about 30 to about 90 minutes, in some cases, the L AMP reaction is performed for at least about 30 minutes, in some cases, the L AMP reaction is performed for up to about 90 minutes, in some cases, the L AMP reaction is performed for about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 45 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 55 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 65 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 75 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 35 minutes to about 40 minutes, about 35 minutes to about 45 minutes, about 35 minutes to about 50 minutes, about 35 minutes to about 55 minutes, about 35 minutes to about 60 minutes, about 35 minutes to about 65 minutes, about 35 minutes to about 70 minutes, about 50 minutes to about 60 minutes, about 50 minutes to about 55 minutes, about 50 minutes to about 60 minutes, about 50 minutes to about 55 minutes, about 60 minutes, about 50 minutes to about 60 minutes, about 60 minutes to about 60 minutes, about 50 minutes to about 60 minutes, about 45 minutes, about 50 minutes to about 60 minutes, about 45 minutes, about 60 minutes to about 60 minutes, about 50 minutes, about 60 minutes to about 55 minutes, about 60 minutes, about 50 minutes to about 45 minutes, about 45 minutes to about 50 minutes, about 45 minutes to about 60 minutes, about 50 minutes, about 45 minutes to about 60 minutes, about 45 minutes, about 60 minutes to about 45 minutes, about 60 minutes, about 45 minutes, about 50 minutes, about 60 minutes to about 60 minutes, about 45 minutes, about 60 minutes to about 60 minutes, about 60 minutes to about 50 minutes, about 60 minutes to about 60 minutes, about 50 minutes, about 60 minutes to about 60 minutes, about 50 minutes, about 60 minutes to about 55 minutes, about 60 minutes to about 50 minutes, about 60 minutes to about 50 minutes to about 60 minutes, about 45 minutes to about 55 minutes, about 60 minutes, about 55 minutes to about 50 minutes to about 60 minutes to about 50 minutes, about 60 minutes to about 60 minutes, about 50 minutes to about 60 minutes to.

In some cases, the Amplification method is Nucleic Acid Sequence-Based Amplification (NASBA). NASBA (also known as 3SR and transcription-mediated Amplification) is an RNA Amplification system Based on isothermal transcription using three enzymes (avian myeloblastosis virus reverse transcriptase, rnase H, and T7 DNA-dependent RNA polymerase) to generate single-stranded RNA in some cases NASBA can be used to amplify DNA Amplification reactions are performed at 41 ℃, maintained at a constant temperature, typically for about 60 to about 90 minutes (see, e.g., Fakruddin, et al, 2012, "Nucleic Acid Based Sequence Amplification (NASBA) reactions and applications," int.j.of L ife Science and Pharma res.2(1): L106-L121, incorporated herein by reference).

In some cases, the NASBA reaction is performed at about 40 ℃ to about 42 ℃. In some cases, the NASBA reaction is performed at 41 ℃. In some cases, the NASBA reaction is performed at up to about 42 ℃. In some cases, the NASBA reaction is performed at about 40 ℃ to about 41 ℃, about 40 ℃ to about 42 ℃, or about 41 ℃ to about 42 ℃. In some cases, the NASBA reaction is performed at about 40 ℃, about 41 ℃, or about 42 ℃.

In some cases, the amplification reaction is performed using NASBA for about 45 to about 120 minutes. In some cases, the NASBA reaction is carried out for about 30 minutes to about 120 minutes. In some cases, the NASBA reaction is carried out for at least about 30 minutes. In some cases, the NASBA reaction is carried out for up to about 120 minutes. In some cases, the NASBA reaction is carried out for up to 180 minutes. In some cases, the NASBA reaction is carried out for about 30 minutes to about 45 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 65 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 75 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 85 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 95 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 120 minutes, about 45 minutes to about 60 minutes, about 45 minutes to about 65 minutes, about 45 minutes to about 70 minutes, about 45 minutes to about 75 minutes, about 45 minutes to about 80 minutes, about 45 minutes to about 85 minutes, about 45 minutes to about 90 minutes, about 45 minutes to about 95 minutes, about 45 minutes to about 100 minutes, about 45 minutes to about 120 minutes, about 60 minutes to about 65 minutes, about 60 minutes to about 70 minutes, about 60 minutes to about 75 minutes, about 60 minutes to about 80 minutes, about 60 minutes to about 80 minutes, About 60 minutes to about 85 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 95 minutes, about 60 minutes to about 100 minutes, about 60 minutes to about 120 minutes, about 65 minutes to about 70 minutes, about 65 minutes to about 75 minutes, about 65 minutes to about 80 minutes, about 65 minutes to about 85 minutes, about 65 minutes to about 90 minutes, about 65 minutes to about 95 minutes, about 65 minutes to about 100 minutes, about 65 minutes to about 120 minutes, about 70 minutes to about 75 minutes, about 70 minutes to about 80 minutes, about 70 minutes to about 85 minutes, about 70 minutes to about 90 minutes, about 70 minutes to about 95 minutes, about 70 minutes to about 100 minutes, about 70 minutes to about 120 minutes, about 75 minutes to about 80 minutes, about 75 minutes to about 85 minutes, about 75 minutes to about 90 minutes, about 75 minutes to about 95 minutes, about 75 minutes to about 100 minutes, about 75 minutes to about 120 minutes, about 75 minutes to about 80 minutes, about 75 minutes to about 85 minutes, about 75 minutes to about 90 minutes, about 75 minutes to about 95 minutes, about 75 minutes to about 100 minutes, about 75 minutes, About 80 minutes to about 85 minutes, about 80 minutes to about 90 minutes, about 80 minutes to about 95 minutes, about 80 minutes to about 100 minutes, about 80 minutes to about 120 minutes, about 85 minutes to about 90 minutes, about 85 minutes to about 95 minutes, about 85 minutes to about 100 minutes, about 85 minutes to about 120 minutes, about 90 minutes to about 95 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 120 minutes, about 95 minutes to about 100 minutes, about 95 minutes to about 120 minutes, or about 100 minutes to about 120 minutes. In some cases, the NASBA reaction is performed for about 30 minutes, about 45 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 120 minutes, about 150 minutes, or about 180 minutes.

In some cases, the amplification method is Strand Displacement Amplification (SDA). SDA is an isothermal amplification method using four different primers. Primers containing restriction sites (recognition sequence for HincII exonuclease) were annealed to the DNA template. Escherichia coli (Escherichia coli) DNA polymerase 1 exonuclease deficient fragment (exo-Klenow) extension primer. Each SDA cycle consisted of: (1) primer binding to displaced target fragment, (2) extension of primer/target complex by exo-Klenow, (3) cleavage of the generated hemi phosphorothioate HincII site, (4) dissociation of HincII from nicks, and (5) nick extension and displacement of the downstream strand by exo-Klenow.

In some cases, the method comprises contacting DNA in the sample with a helicase. In some cases, the amplification method is Helicase Dependent Amplification (HDA). HDA is an isothermal reaction because helicase is used to denature DNA, rather than heat.

In some cases, the amplification method is Multiple Displacement Amplification (MDA). MDA is an isothermal strand displacement method based on the use of a DNA derived from a bacteriophageThe highly processed and strand-displaced DNA polymerase of (1), in combination with the modified random primers, amplifies the entire genome with high fidelity. It has been developed to amplify all DNA in a sample from a very small amount of starting material. In MDAIn (2), a DNA polymerase is incubated with dNTPs, random hexamers and denatured template DNA at 30 ℃ for 16 to 18 hoursAnd the enzyme must be inactivated at high temperature (65 ℃) for 10 minutes. No repeated cycles are required, but a short initial denaturation step, amplification step and final inactivation of the enzyme are required.

In some cases, the amplification method is Rolling Circle Amplification (RCA). RCA is an isothermal nucleic acid amplification method that allows amplification of probe DNA sequences at a single temperature (typically about 30 ℃) 10⁹More than twice. Multiple rounds of isothermal enzymatic synthesis fromA DNA polymerase that extends the loop-hybridizing primer by progressing around the circular DNA probe. In some cases, the amplification reaction is performed using RCA at about 28 ℃ to about 32 ℃.

Additional amplification methods that can incorporate the devices and methods disclosed herein can be found in the art. Ideally, the amplification method is isothermal and rapid relative to conventional PCR. In some cases, amplification comprises performing an exponential amplification reaction (EXPAR), which is an isothermal molecular chain reaction, as the product of one reaction catalyzes a further reaction that produces the same product. In some cases, amplification occurs in the presence of an endonuclease. The endonuclease may be a nicking endonuclease. See, e.g., Wu et al, "aligned-medial Cleavase of Nucleic Acids," Chemical Science (2018). In some cases, the amplification does not require initial thermal denaturation of the target DNA. See, for example, Toley et al, "Isothermal strand and displacement amplification (iSDA): a Rapid and sensory methods for point-of-care diagnostics," The analysis (2015). Pulsed controlled amplification in the ultra-fast amplification method was developed by GNA Biosolutions GmbH.

Sequencing

In some cases, the methods disclosed herein comprise sequencing a nucleic acid. The nucleic acid can be a nucleic acid disclosed herein, such as a labeled nucleic acid, an amplified nucleic acid, a cell-free fetal nucleic acid, a nucleic acid having a sequence corresponding to a target chromosome region, a nucleic acid having a sequence corresponding to a non-target chromosome, or a combination thereof. In some cases, the nucleic acid is DNA. In some cases, the nucleic acid is RNA. In some cases, the nucleic acid comprises DNA. In some cases, the nucleic acid comprises RNA. In some cases, the method comprises bisulfite sequencing to detect epigenetic modifications.

In some cases, sequencing comprises targeted sequencing. In some cases, sequencing comprises whole genome sequencing. In some cases, sequencing includes targeted sequencing and whole genome sequencing. In some cases, whole genome sequencing comprises massively parallel sequencing, also referred to in the art as next generation sequencing or second generation sequencing. In some cases, whole genome sequencing comprises random massively parallel sequencing. In some cases, sequencing comprises random massively parallel sequencing of target regions captured from an entire genomic library.

In some cases, a method comprises sequencing an amplified nucleic acid disclosed herein. In some cases, the amplified nucleic acid is produced by targeted amplification (e.g., using primers specific for the target sequence of interest). In some cases, the amplified nucleic acid is produced by non-targeted amplification (e.g., using random oligonucleotide primers). In some cases, the method comprises sequencing the amplified nucleic acids, wherein the sequencing comprises massively parallel sequencing.

Library preparation

In some cases, the methods disclosed herein comprise modifying nucleic acids in a biological sample to generate a library of nucleic acids for detection. In some cases, a method includes modifying a nucleic acid for nucleic acid sequencing. In some cases, the method comprises modifying the nucleic acid for detection, wherein detecting does not comprise nucleic acid sequencing. In some cases, the method comprises modifying the nucleic acid for detection, wherein detecting comprises counting labeled nucleic acids based on the occurrence of label detection. In some cases, the methods disclosed herein comprise modifying nucleic acids in a biological sample to generate a nucleic acid library, wherein the methods comprise amplifying the nucleic acids. In some cases, the modification occurs prior to amplification. In some cases, the modification occurs after amplification.

In some cases, the methods disclosed herein include preparing a non-selective library (e.g., incorporating all or many of the available cfDNA or DNA analyte fragments into the library preparation). In other instances, the methods disclosed herein comprise preparing a targeted or selective library, wherein the nucleic acid of interest is selected prior to or during library preparation. By way of non-limiting example, a Y chromosome-specific library (e.g., a DNA fragment having a sequence found only on the Y chromosome) can be prepared. Similarly, an X chromosome-specific library, an autosomal specific library, or a custom-made library having a particular sequence, gene, or region of a gene of interest can be prepared.

In some cases, modifying the nucleic acid comprises repairing the ends of the nucleic acid that are nucleic acid fragments. By way of non-limiting example, repairing the terminus can include restoring a 5 'phosphate group, a 3' hydroxyl group, or a combination thereof to the nucleic acid. In some cases, repairing may include removing overhangs. In some cases, repairing may include filling the overhangs with complementary nucleotides.

In some cases, modifying nucleic acids to make libraries includes the use of adapters. Adapters may also be referred to herein as sequencing adapters. In some cases, the adapters facilitate sequencing. Typically, the adaptor comprises an oligonucleotide. By way of non-limiting example, the adapter can simplify other steps in the method, such as amplification, purification, and sequencing, because it is a universal sequence for multiple (e.g., not all) nucleic acids in the modified sample. In some cases, modifying the nucleic acid comprises ligating an adaptor to the nucleic acid. The connection may comprise a flat end connection. In some cases, modifying the nucleic acid comprises hybridizing an adaptor to the nucleic acid.

In some cases, modifying the nucleic acid to make the library includes the use of a tag. The label may also be referred to herein as a barcode. In some cases, the methods disclosed herein comprise modifying a nucleic acid with a tag corresponding to a chromosomal region of interest. In some cases, the methods disclosed herein comprise modifying a nucleic acid with a tag that is specific for a chromosomal region that is not of interest. In some cases, the methods disclosed herein comprise modifying a first portion of the nucleic acid with a first tag corresponding to at least one chromosomal region of interest, and modifying a second portion of the nucleic acid with a second tag corresponding to at least one chromosomal region of non-interest. In some cases, modifying the nucleic acid comprises attaching a tag to the nucleic acid. The connection may comprise a flat end connection. In some cases, modifying the nucleic acid comprises hybridizing a tag to the nucleic acid. In some cases, the tag comprises an oligonucleotide. In some cases, the tag comprises a non-oligonucleotide marker or label that can be detected by means other than nucleic acid analysis. By way of non-limiting example, non-oligonucleotide markers or labels may include fluorescent molecules, nanoparticles, dyes, peptides, or other small molecules that are detectable/quantifiable.

In some cases, modifying nucleic acids to prepare libraries includes the use of sample indices, also referred to herein simply as indices. By way of non-limiting example, the index may include oligonucleotides, small molecules, nanoparticles, peptides, fluorescent molecules, dyes, or other detectable/quantifiable moieties. In some cases, a first set of nucleic acids from a first biological sample is labeled with a first index and a first set of nucleic acids from a first biological sample is labeled with a second index, wherein the first index and the second index are different. Thus, when analyzing multiple samples at a time, the multiple indices allow for distinguishing nucleic acids from multiple samples. In some cases, the methods disclose amplifying a nucleic acid, wherein the oligonucleotide primers used to amplify the nucleic acid comprise an index.

In some cases, the method comprises detecting an amplification product, wherein the amplification product is produced by amplifying at least a portion of a target chromosome disclosed herein or a fragment thereof. The portion or fragment of the target chromosome may comprise at least 5 nucleotides. The portion or fragment of the target chromosome may comprise at least about 10 nucleotides. The portion or fragment of the target chromosome may comprise at least about 15 nucleotides. In some cases, detecting an amplification product disclosed herein does not comprise tagging or labeling the amplification product. In some cases, the method detects the amplification product based on the amount of the amplification product. For example, the method can detect an increase in the amount of double-stranded DNA in a sample. In some cases, detecting the amplification product is based at least in part on its size. In some cases, the amplification product has a length of about 50 base pairs to about 500 base pairs.

In some cases, detecting the amplification product comprises contacting the amplification product with a tag. In some cases, the tag comprises a sequence that is complementary to a sequence of the amplification product. In some cases, the tag does not comprise a sequence that is complementary to the sequence of the amplification product. Non-limiting examples of labels are described in the foregoing and following disclosures.

In some cases, detecting the labeled or unlabeled amplification product comprises subjecting the amplification product to a signal detector or assay assembly of a device, system, or kit disclosed herein. In some cases, the methods comprise amplification and detection on an assay assembly of a device, system, or kit disclosed herein. In some cases, the assay assembly comprises amplification reagents.

In some aspects, the methods disclosed herein comprise: obtaining a fluid sample from a female pregnant subject; contacting at least one circulating cell-free nucleic acid in a sample with at least one tag to produce labeled nucleic acids, wherein the circulating cell-free nucleic acid comprises a sequence corresponding to the Y chromosome; and detecting the labeled nucleic acid. In some cases, the method further comprises amplifying the labeled nucleic acids to produce a plurality of labeled nucleic acids and detecting the plurality of labeled nucleic acids. In some cases, the tag enables capture of circulating cell-free nucleic acids or amplification products thereof. In some cases, the tag enables detection of circulating cell-free nucleic acids or amplification products thereof. In some cases, the circulating cell-free nucleic acid is double-stranded DNA, and the method comprises separating at least a portion of the double-stranded DNA to produce single-stranded DNA prior to contacting at least one circulating cell-free nucleic acid in the sample with the at least one tag. In some cases, the separating comprises applying heat to the cell-free nucleic acid. In some cases, the isolating comprises applying the enzyme to cell-free nucleic acids. In some cases, the tag comprises an oligonucleotide. In some cases, the tag comprises a peptide or protein. In some cases, the tag comprises a small molecule. Small molecules may be organic or inorganic.

In some cases, the methods disclosed herein comprise contacting at least one nucleic acid in a biological sample with a labeled oligonucleotide primer. In some cases, the labeled oligonucleotide primer comprises an oligonucleotide primer and an oligonucleotide tag. In some cases, the labeled oligonucleotide primer comprises an oligonucleotide primer and a tag, wherein the tag does not comprise a nucleotide. In some cases, the labeled oligonucleotide primer comprises an oligonucleotide primer and a tag, wherein the tag does not comprise an oligonucleotide. In some cases, the labeled oligonucleotide primer comprises an oligonucleotide primer and a peptide tag. In some cases, the labeled oligonucleotide primer comprises an oligonucleotide primer and a small molecule tag. In some aspects, disclosed herein are methods comprising: obtaining a fluid sample from a female pregnant subject; contacting at least one circulating cell-free nucleic acid in the sample with at least one labeled oligonucleotide primer, wherein the circulating cell-free nucleic acid comprises a sequence corresponding to the Y chromosome; amplifying the circulating cell-free nucleic acid by contacting the extended circulating cell-free nucleic acid with a polymerase and free nucleotides, thereby producing a labeled amplification product; and detecting the tag portion of the labeled amplification product. In some cases, the circulating cell-free nucleic acid is double-stranded DNA, and the method comprises separating at least a portion of the double-stranded DNA to produce single-stranded DNA prior to contacting at least one circulating cell-free nucleic acid in the sample with at least one labeled oligonucleotide primer. In some cases, the separating comprises applying heat to the cell-free nucleic acid. In some cases, the isolating comprises applying the enzyme to cell-free nucleic acids.

Alternatively or additionally, methods include contacting the sample with an oligonucleotide tag that includes a sequence that corresponds to a sequence on the Y chromosome and at least one oligonucleotide primer that includes a sequence that corresponds to a sequence on the Y chromosome, wherein the oligonucleotide tag is separate from the oligonucleotide primer.

In some cases, the methods disclosed herein include the use of multiple tags, thereby increasing at least one of the accuracy of the method, the speed of the method, and the information obtained by the method. In some cases, the methods disclosed herein include the use of multiple tags, thereby reducing the sample volume required to obtain reliable results. In some cases, the methods disclosed herein comprise contacting nucleic acids in a biological sample having a plurality of tags with a plurality of regions of the Y chromosome. In some cases, the methods disclosed herein comprise contacting nucleic acids in a biological sample having a plurality of tags with a plurality of regions of the Y chromosome, thereby labeling the entire Y chromosome. In some cases, the methods disclosed herein comprise contacting nucleic acids in a biological sample having a plurality of tags with a plurality of regions of the Y chromosome, thereby labeling a percentage of the Y chromosome. In some cases, the percentage is from about 1% to about 99%. In some cases, the percentage is about 10% to 99%. In some cases, the percentage is about 10% to about 99%. In some cases, the percentage is about 20% to 99%. In some cases, the percentage is about 30% to about 99%. In some cases, the percentage is about 40% to about 99%. In some cases, the percentage is about 50% to about 99%. In some cases, the percentage is about 60% to about 99%. In some cases, the percentage is about 70% to about 99%. In some cases, the percentage is about 80% to about 99%. In some cases, the percentage is about 90% to about 99%. In some cases, the percentage is from about 1% to about 99%. In some cases, the percentage is about 10% to about 20%. In some cases, the percentage is about 10% to about 30%. In some cases, the percentage is about 10% to about 40%. In some cases, the percentage is about 10% to about 50%. In some cases, the percentage is about 10% to about 60%. In some cases, the percentage is about 10% to about 70%. In some cases, the percentage is about 60% to about 99%. In some cases, the percentage is about 10% to about 80%. In some cases, the percentage is about 80% to about 99%. In some cases, the percentage is about 10% to about 90%.

In some cases, the plurality of tags includes at least one capture tag. In some cases, the plurality of tags includes at least one detection tag. In some cases, the plurality of tags includes a combination of at least one capture tag and at least one detection tag. In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag comprises a first oligonucleotide that is complementary to a sense strand of the circulating cell-free nucleic acid and the second capture tag comprises a second oligonucleotide that is complementary to an antisense strand of the circulating cell-free nucleic acid. In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag carries the same label as the second tag. In some cases, the method comprises contacting at least one circulating cell-free nucleic acid in the sample with a first tag and a second tag, wherein the first tag carries a label that is different from the second tag.

Detecting and determining genetic information

Generally, the methods disclosed herein comprise detecting a biomarker, an analyte, or a modified form thereof. The method may comprise detecting a plurality of analytes sharing a common characteristic. In some cases, the analyte is a nucleic acid, the common characteristic is a sequence, and detecting comprises sequencing the nucleic acid or an amplicon thereof. In some cases, the common characteristic is an epigenetic state, such as a methylation state. In some cases, the method comprises detecting a label or signal on the target analyte.

In some cases, a method comprises detecting a nucleic acid. In some cases, the method comprises detecting cell-free nucleic acid. In some cases, the method comprises detecting a tag that has been linked or hybridized to a nucleic acid. In some cases, the method comprises detecting an amplicon of the nucleic acid. Alternatively or additionally, the method comprises detecting a non-nucleic acid component. By way of non-limiting example, the non-nucleic acid component can be selected from the group consisting of proteins, peptides, lipids, fatty acids, sterols, carbohydrates, viral components, microbial components, and combinations thereof. In the case of viral or microbial components, the method may comprise releasing, purifying and/or amplifying nucleic acid from the virus or bacteria prior to detection.

The method may include detecting a detectable label or detectable signal of a binding moiety (e.g., a small molecule, peptide, aptamer, antibody, or antigen binding fragment thereof) that binds to a nucleic acid or non-nucleic acid component.

The methods disclosed herein may include detecting and/or monitoring epigenetic changes from a small biological sample. The method can include detecting an epigenetic state of a plurality of cell-free DNA fragments from one or more target regions. The method can include detecting an epigenetic state of a plurality of cytosines from one or more target regions that are sufficiently distant from each other to be present on an isolated cell-free DNA fragment. For example, assessing cytosine methylation in circulating cell-free DNA from the INS1 locus may be indicative of B cell degradation found in autoimmune type 1 diabetes and may therefore serve as a biomarker of risk for type 1 diabetes. Similarly, the cytosine methylation state of genes encoding Myelin Oligodendrocyte Glycoprotein (MOG), Myelin Basic Protein (MBP), macroglobulinemia, waldenstrom susceptibility protein 1(WM1), or a combination thereof, can be used as a non-invasive biomarker for Multiple Sclerosis (MS). As another example, assessing cytosine methylation in CpG islands of the AOX1 gene promoter can aid in the diagnosis of prostate cancer (PCa) and can allow monitoring of progression and treatment success. The AOX1 gene is located on chromosome 2. The promoter CpG island is located between base positions 200,585,800 and 200,586,350 and spans about 500 bp. It contains at least 34 CpG nucleotides, all of which are hypermethylated in prostate cancer but unmethylated in normal samples. These specific CpG nucleotides in circulating cell-free DNA can be analyzed as groups, subgroups, or at a single level using the devices, systems, and methods described herein.

As described herein, detection can include amplification, for example, amplification can include qPCR, wherein a signal is generated based on the presence or absence of a target analyte in some cases, methods include detecting nucleic acid amplification products from L AMP reactions by detecting the turbidity in L AMP reaction vessels (Mori et al (2004)59:145-147 call a real-time turbidimeter). L AMP rapidly produces large quantities of specific amplicons while forming magnesium pyrophosphate precipitates.

In some cases, a method comprises detecting an amplification product, wherein the amplification product is produced by amplifying at least a portion of a target region. The target region may comprise at least 5 nucleotides. The target region may comprise at least about 10 nucleotides. The target region may comprise at least about 15 nucleotides. In some cases, detecting an amplification product disclosed herein does not comprise tagging or labeling the amplification product. In some cases, the method detects the amplification product based on the amount of the amplification product. For example, the method can detect an increase in the amount of double-stranded DNA in a sample. In some cases, detecting the amplification product is based at least in part on its size. In some cases, the amplification product has a length of about 50 base pairs to about 250 base pairs. In some cases, the amplification product has a length of about 50 base pairs to about 300 base pairs. In some cases, the amplification product has a length of about 50 base pairs to about 400 base pairs. In some cases, the amplification product has a length of about 50 base pairs to about 500 base pairs. In some cases, the amplification product has a length of about 50 base pairs to about 1000 base pairs.

In some cases, detecting the amplification product comprises contacting the amplification product with a tag. In some cases, the tag comprises a sequence that is complementary to a sequence of the amplification product. In some cases, the tag does not comprise a sequence that is complementary to the sequence of the amplification product. Non-limiting examples of labels are described in the foregoing and following disclosures.

In some cases, detecting the labeled or unlabeled amplification product comprises subjecting the amplification product to a signal detector or assay assembly of a device, system, or kit disclosed herein. In some cases, the methods comprise amplification and detection on an assay assembly of a device, system, or kit disclosed herein. In some cases, the assay assembly comprises amplification reagents.

In some cases, detecting the nucleic acid does not include amplifying the nucleic acid or a portion thereof. In some cases, detecting the nucleic acid does not include sequencing the nucleic acid or a portion thereof. In some cases, detecting the nucleic acid does not include sequencing or amplifying the nucleic acid or portion thereof. For example, in some cases, a nucleic acid can be labeled with a labeled probe, and detection of the labeled probe is sufficient to detect the absence, presence, or quantity of the nucleic acid. Accordingly, the apparatus and systems disclosed herein capable of performing such methods may not comprise amplification reagents, sequencing devices, or combinations thereof.

In some cases, detecting comprises subjecting the biomarker to a lateral flow assay. The detecting may further comprise applying an instrument or reagent to the lateral flow assay to control the flow of the biological sample, solution, or combination thereof through the lateral flow assay. In some cases, the instrument is a vacuum, pump, pipette, or a combination thereof.

In some cases, the method includes detecting a highly repetitive region (e.g., HRR). A highly repetitive region may be a region comprising at least two sequences that are at least 50% identical. In some cases, the highly repetitive region comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sequences that are at least 50% identical. In some cases, at least two regions are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% identical. In some cases, the at least two sequences should be sufficiently far apart that they occur in two separate cell-free DNA fragments. In some cases, the at least two sequences are separated by at least one nucleotide. In some cases, the at least two sequences are separated by at least two nucleotides. In some cases, the at least two sequences are separated by at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 nucleotides. In some cases, the at least two sequences are separated by up to about 200 nucleotides. By way of non-limiting example, the HRR may be a highly repetitive Y chromosome region (HRYR).

In some cases, the method includes detecting multiple copies of the sequence of interest. In some cases, the copy number is from 1 to about 50,000. In some cases, the copy number is from about 1 to about 50. In some cases, the copy number is from 1 to about 500. In some cases, the copy number is from 1 to about 1,000. In some cases, the copy number is from 1 to about 2,000. In some cases, the copy number is from 1 to about 5,000. In some cases, the copy number is less than about 10,000. In some cases, the copy number is less than about 5,000. In some cases, the copy number is from 4 to about 20,000. In some cases, the copy number is from 4 to about 10,000. In some cases, the copy number is from 4 to about 5,000. In some cases, the copy number is from 4 to about 1,000. In some cases, the copy number is less than about 1,000. In some cases, the copy number is less than about 500. In some cases, the copy number is less than about 200. In some cases, the copy number is less than about 100. In some cases, the copy number is less than about 50. In some cases, the copy number is less than about 40. In some cases, the copy number is less than about 20. In some cases, the copy number is at least 1. In some cases, the copy number is at least 2. In some cases, the copy number is at least 4. In some cases, the copy number is at least 5. In some cases, the copy number is at least 10. In some cases, the sequence of interest is a sequence specific for the Y chromosome. By way of non-limiting example, a method may include detecting a male fetus as long as a copy of the Y chromosome region or a fragment of the Y chromosome containing the sequence of interest is present in the sample.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid corresponding to a Y chromosome region. In some cases, the cell-free nucleic acid comprises a sequence found on the Y chromosome. In some cases, the cell-free nucleic acid comprises a sequence found only on the Y chromosome. In some cases, the cell-free nucleic acid comprises a sequence not found on the X chromosome or any autosome. In some cases, a Y chromosome sequence is a sequence that occurs more than once on the Y chromosome. In some cases, the Y chromosome sequence is a first sequence that is a homolog of a second sequence, wherein the second sequence is also found on the Y chromosome. In some cases, the first sequence is at least 80% identical to the second sequence. In some cases, the first sequence is at least 85% identical to the second sequence. In some cases, the first sequence is at least 90% identical to the second sequence. In some cases, the first sequence is at least 95% identical to the second sequence. In some cases, the first sequence and the second sequence are at least 15 nucleotides in length. In some cases, the first sequence and the second sequence are at least 25 nucleotides in length. In some cases, the first sequence and the second sequence are at least 50 nucleotides in length. In some cases, the first sequence and the second sequence are at least 100 nucleotides in length.

In some cases, the method comprises detecting a nucleic acid corresponding to a region of the Y chromosome, or a portion thereof, comprising a sequence that is present more than once on the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20500000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 20500000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20000000 and position 20250000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20250000 and position 20500000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20500000 and position 20750000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20750000 and position 21000000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20080000 and position 20400000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20082000 and position 20351000 of the Y chromosome. In some cases, the Y chromosome region is located between position 20082183 and position 20350897 of the Y chromosome. In some cases, the correspondence is 100% identical. In some cases, the correspondence is at least 99% identical. In some cases, the correspondence is at least 98% identical. In some cases, the correspondence is at least 95% identical. In some cases, the correspondence is at least 90% identical.

In some cases, the methods disclosed herein comprise detecting or quantifying a circulating cell-free nucleic acid comprising a sequence corresponding to a Y chromosome region, or a portion thereof, located between position 20000000 and position 21000000 of the Y chromosome, wherein the Y chromosome region has a given length. In some cases, the Y chromosome region is about 10 nucleotides to about 1,000,000 nucleotides in length. In some cases, the Y chromosome region is about 10 nucleotides to about 500,000 nucleotides in length. In some cases, the Y chromosome region is from about 10 nucleotides to about 300,000 nucleotides in length. In some cases, the Y chromosome region is about 100 nucleotides to about 1,000,000 nucleotides in length. In some cases, the Y chromosome region is about 100 nucleotides to about 500,000 nucleotides in length. In some cases, the Y chromosome region is about 100 nucleotides to about 300,000 base pairs in length. In some cases, the Y chromosome region is about 1000 nucleotides to about 1,000,000 nucleotides in length. In some cases, the Y chromosome region is about 1000 nucleotides to about 500,000 nucleotides in length. In some cases, the Y chromosome region is about 1000 nucleotides to about 300,000 nucleotides in length. In some cases, the Y chromosome region is about 10,000 nucleotides to about 1,000,000 nucleotides in length. In some cases, the Y chromosome region is about 10,000 nucleotides to about 500,000 nucleotides in length. In some cases, the Y chromosome region is about 10,000 nucleotides to about 300,000 nucleotides in length. In some cases, the Y chromosome region is about 300,000 nucleotides in length.

In some cases, the methods disclosed herein comprise detecting or quantifying a circulating cell-free nucleic acid comprising a sequence corresponding to a Y chromosome region, or a portion thereof, located between position 20000000 and position 21000000 of the Y chromosome, wherein the sequence is of a given length. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a Y chromosome region. In some cases, the sequence is from about 10 nucleotides to about 1,000 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 500 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 400 nucleotides in length. In some cases, the sequence is from about 10 nucleotides to about 300 nucleotides in length. In some cases, the sequence is from about 50 nucleotides to about 1000 nucleotides in length. In some cases, the sequence is from about 50 nucleotides to about 500 nucleotides in length.

In some cases, the methods disclosed herein comprise detecting or quantifying a circulating cell-free nucleic acid comprising a sequence corresponding to a Y chromosome region or a portion thereof, wherein the portion thereof has a given length. In some cases, the portion thereof is from about 10 nucleotides to about 100 nucleotides in length. In some cases, the portion thereof is from about 100 nucleotides to about 1000 nucleotides in length. In some cases, the portion thereof is from about 1000 nucleotides to about 10,000 nucleotides in length. In some cases, the portion thereof is from about 10,000 nucleotides to about 100,000 nucleotides in length.

In some cases, the methods disclosed herein comprise detecting at least one circulating cell-free nucleic acid comprising a sequence corresponding to a Y chromosome subregion of a Y chromosome region disclosed herein. In some cases, the sub-region is represented by a sequence that is present more than once in the Y chromosome region. In some cases, the correspondence is 100% identical. In some cases, the correspondence is at least 99% identical. In some cases, the correspondence is at least 98% identical. In some cases, the correspondence is at least 95% identical. In some cases, the correspondence is at least 90% identical.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 20350799 and end position 20350897 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 10 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 100 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 200 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 500 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350799 and the end position 20350897 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 56673250 and end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least 10 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least 50 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 10 to at least about 1000 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 500 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome. In some cases, the sequence corresponds to at least about 50 to at least about 150 nucleotides of a sub-region of the Y chromosome between the start position 56673250 and the end position 56771489 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 20349236 and end position 20349318 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 10 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 100 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 200 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 500 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20349236 and the end position 20349318 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 20350231 and end position 20350323 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 10 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 100 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 200 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 500 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350231 and the end position 20350323 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 20350601 and end position 20350699 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 10 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 100 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 200 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 500 nucleotides corresponding to a sub-region of the Y chromosome between the start position 20350601 and the end position 20350699 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome between start position 20082183 and end position 20082281 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 10 nucleotides corresponding to a sub-region of the Y chromosome between start position 20082183 and end position 20082281 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 100 nucleotides corresponding to a sub-region of the Y chromosome between start position 20082183 and end position 20082281 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 200 nucleotides corresponding to a sub-region of the Y chromosome between start position 20082183 and end position 20082281 of the Y chromosome. In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence of at least 500 nucleotides corresponding to a sub-region of the Y chromosome between start position 20082183 and end position 20082281 of the Y chromosome.

In some cases, the methods disclosed herein comprise detecting a circulating cell-free nucleic acid comprising a sequence corresponding to a subregion of the Y chromosome, wherein the sequence is selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 60% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 65% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 70% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 75% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 80% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 85% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 98% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is at least 99% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192. In some cases, the sequence is 100% identical to a sequence selected from the group consisting of SEQ ID Nos. 1-5, 30-34, and 141-192.

In some cases, the method comprises detecting a nucleic acid corresponding to a Y chromosome sequence having a homolog or copy of a sequence present in the Y chromosome gene on the Y chromosome, in some cases the Y chromosome sequence is located in a repeat region of the Y chromosome, in some cases the repeat region comprises a pseudogene, a nearly exact copy of the gene (> 90% homology when aligned for maximum homology), an intergenic region, or a microsatellite repeat or an identifiable portion thereof (e.g., at least 10 nucleotides). non-limiting examples of Y chromosome genes are Y-linked testis-specific protein 1(TSPY1) (alias DYS14), Y-linked testis-specific protein 2(TSPY2), DYZ1, Y-linked testis-specific transcript 22(TTTY22), sex determining region Y (SRY), Y4Y-linked protein 1(RPS4Y1), Y-linked zinc finger protein (Y), TGIF 2L Y. in some cases, the Y chromosome sequence comprises at least 100 contiguous nucleotides selected from the same as the sequence of SEQ ID No. 1, Y chromosome 1-192, Y chromosome sequence 20-20, at least 10 contiguous nucleotides selected from the same as the sequence of SEQ ID No. 20, 20-20, at least 10 contiguous nucleotides contained from the same as the sequence of Y chromosome 20, in cases where the Y chromosome sequence comprises at least 100 contiguous nucleotides 20, at least 10 contiguous nucleotides contained from the same as the same contiguous nucleotides contained from the same as the sequence of SEQ ID No. 20, 10 contiguous nucleotides contained from the same as the same nucleotide sequence of SEQ ID No. 20, 10, the same contiguous nucleotides contained from the same contiguous nucleotide sequence of SEQ ID, the same as the same contiguous nucleotide sequence of the same nucleotide sequence of SEQ ID No. 20, SEQ ID No. 20, SEQ ID No..

The detection may include viewing an interface of the device or system disclosed herein, wherein the test results are displayed. Detection may include viewing a color appearance or a fluorescent signal on the lateral flow device. Detecting may include receiving the test results on the devices disclosed herein. Detecting may include receiving the test results on a mobile device, computer, notebook, or other electronic device in communication with the devices of the system disclosed herein.

In general, the methods, kits, systems, and devices disclosed herein are capable of providing genetic information (e.g., fetal gender) in a short period of time. In some cases, the methods disclosed herein can be performed in less than about 1 minute. In some cases, the methods disclosed herein can be performed in less than about 2 minutes. In some cases, the methods disclosed herein can be performed in less than about 5 minutes. In some cases, the methods disclosed herein can be performed in less than about 10 minutes. In some cases, the methods disclosed herein can be performed in less than about 15 minutes. In some cases, the methods disclosed herein can be performed in less than about 20 minutes. In some cases, the methods disclosed herein can be performed in less than about 30 minutes. In some cases, the methods disclosed herein can be performed in less than about 45 minutes. In some cases, the methods disclosed herein can be performed in less than about 60 minutes. In some cases, the methods disclosed herein can be performed in less than about 90 minutes. In some cases, the methods disclosed herein can be performed in less than about 2 hours. In some cases, the methods disclosed herein can be performed in less than about 3 hours. In some cases, the methods disclosed herein can be performed in less than about 4 hours.

The use of the methods, kits, systems and devices disclosed herein generally does not require any technical training. For example, the kits, systems, and devices disclosed herein may be used by pregnant subjects in their homes without the assistance of a technician or medical provider. In some cases, the methods disclosed herein may be performed by a user without medical or technical training. In some cases, the methods, kits, systems, and devices disclosed herein only require the user to add a biological sample to the system or device, optionally turn on the power of the system or device, and view the results to obtain genetic information.

Aspects relating to devices, systems, kits and methods

The following aspects relate to the devices, systems, kits, and methods disclosed herein. The devices, systems, kits, and methods disclosed herein are generally designed for processing and analyzing biomarkers and nucleic acids in biological, plant, and environmental samples of animal subjects. The following description of biological samples, cell-free nucleic acids, and subjects can help understand the utility of the devices, systems, kits, and methods disclosed herein.

Biological sample

Disclosed herein are devices, systems, kits, and methods for analyzing biomarkers and nucleic acids in biological samples. Typically, biological samples include animal samples, plant samples, and environmental samples. Non-limiting examples of animal samples are blood and urine. Non-limiting examples of plant samples are leafy material and seeds. Non-limiting examples of environmental samples are water samples, treated water, industrial waste, soil samples, food samples in a body of water (e.g., ocean, lake, river, stream). In some cases, the biological sample must be prepared in the form of a fluid solution before it can be used by the devices, systems, kits, or methods disclosed herein.

In some cases, the biological sample is a biological fluid sample. Non-limiting examples of biological fluid samples include samples of whole blood, plasma, serum, saliva, urine, sweat, tears, rectal discharge, cerebrospinal fluid, lymph, synovial fluid, interstitial fluid, and vaginal fluid. In some cases, the biological sample comprises whole blood. In contrast to plasma, whole blood requires little processing. There may be a filtration step to remove some debris from the blood sample without separating red blood cells from white blood cells. In some cases, the biological sample is a swab, such as a cheek swab or a vaginal swab.

The biological samples described herein include biological fluids that are substantially free of cells, or can be modified to be cell-free biological fluids. For example, cell-free nucleic acids can circulate in the bloodstream of a subject, and thus detection reagents can be used to detect or quantify markers in a blood or serum sample from a subject. Unless otherwise indicated, the terms "plasma" and "serum" are used interchangeably herein. However, in some cases they are included in a single sample species list to indicate that both descriptions are included in the specification or claims.

In some cases, the devices, systems, kits, and methods disclosed herein are capable of removing cells from a biological sample. The resulting sample may be referred to as a cell removal sample. The cell-depleted sample can have at least 95% fewer complete, intact cells than the biological sample. The cell-depleted sample can have at least 90% fewer complete, intact cells than the biological sample. The cell-depleted sample can have at least 80% fewer complete, intact cells than the biological sample. The cell-depleted sample can have at least about 75%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, or at least about 25% fewer complete, intact cells than the biological sample. The cell-depleted sample may be completely free of any complete, intact cells.

In some cases, the biological sample comprises capillary blood. In some cases, the biological sample comprises venous blood. Blood obtained from capillaries (e.g., blood vessels at the ends such as fingers, toes, etc.) may be referred to herein as "capillary blood. Blood obtained from veins (e.g., arms, middle of the hand) may be referred to herein as "venous blood". Common veins used for venipuncture to obtain venous blood are the median elbow vein, the cephalic vein, the basilic vein and the dorsal metacarpal vein. In some cases, the biological sample consists essentially of capillary blood. In some cases, the biological sample consists of capillary blood. In some embodiments, the biological sample does not comprise venous blood. In some cases, the biological sample comprises plasma. In some cases, the biological sample consists essentially of plasma. In some cases, the biological sample consists of plasma. In some cases, the biological sample comprises serum. In some cases, the biological sample consists essentially of serum. In some cases, the biological sample consists of serum. In some cases, the biological sample comprises urine. In some cases, the biological sample consists essentially of urine. In some cases, the biological sample consists of urine. In some cases, the biological sample comprises saliva. In some cases, the biological sample consists essentially of saliva. In some cases, the biological sample consists of saliva. In some cases, the biological fluid comprises vaginal fluid. In some cases, the biological fluid consists essentially of vaginal fluid. In some cases, the biological fluid consists of vaginal fluid. In some cases, vaginal fluid is obtained by vaginal swabbing of a pregnant subject. In some cases, the biological sample comprises interstitial fluid. In some cases, the biological sample consists essentially of interstitial fluid. In some cases, the biological sample consists of interstitial fluid.

In some cases, the biological sample is whole blood. In general, the devices, systems, kits, and methods disclosed herein are capable of analyzing cell-free nucleic acids from very small whole blood samples. In some cases, a smaller whole blood sample may be obtained with a finger prick, such as with a lancet or pin/needle. In some cases, smaller whole blood samples may be obtained under phlebotomy conditions. In some cases, the devices, systems, kits, and methods disclosed herein are capable of analyzing cell-free nucleic acids in whole blood without separating the whole blood into blood fractions (serum, plasma, cell fractions).

In some cases, the devices, systems, kits, and methods disclosed herein require at least about 20 μ L blood to provide test results with at least about 95% confidence or accuracy the devices, systems, and kits disclosed herein require at least about 30 μ L blood to provide test results with at least about 95% confidence or accuracy the devices, systems, and kits disclosed herein require at least about 40 μ L blood to provide test results with at least about 95% confidence or accuracy the devices, systems, and kits disclosed herein require at least about 50 μ L blood to provide test results with at least about 95% confidence or accuracy the devices, systems, and kits disclosed herein require at least about 60 μ L blood to provide test results with at least about 95% confidence or accuracy the devices, in some cases, the devices, systems, and kits disclosed herein require at least about 70 μ 582 confidence to provide test results with at least about 95% confidence or accuracy the devices, about 99 μ 465 confidence, about 99, or about 99 μ 465, about 99, or 99 μ 465, about 99, or 99, about 99, or 99, about 99 μ 465, about 99, or about 99, and about 99, about 95, about 99, about 95, about 99, and about 99, about 95, and about 99, about 95, about 99, and about 99, about 95, about 99, about 95, about 99, and about 99, about, and about 95, about 99, about 95.

In some cases, the biological sample is plasma, the plasma constitutes about 55% of whole blood, in some cases, the devices, systems, kits, and methods disclosed herein require at least about 10 μ L plasma to provide test results with at least about 95% confidence or accuracy, in some cases, the devices, systems, and kits disclosed herein require at least about 20 μ L plasma to provide test results with at least about 95% confidence or accuracy, in some cases, the devices, systems, and kits disclosed herein require at least about 30 μ L plasma to provide test results with at least about 95% confidence or accuracy, in some cases, the devices, systems, and kits disclosed herein require at least about 40 μ L plasma to provide test results with at least about 95% confidence or accuracy, in some cases, the devices, systems, and kits disclosed herein require at least about 50 μ L2 to provide test results with at least about 95% confidence or accuracy, in some cases, the devices, systems, and kits require at least about 50 μ L to provide test results with at least about 95% confidence or about 99% confidence, the devices, about 10 μ L, the devices, about 99% confidence, about 99%, or about 10 μ L% to provide test results with at least about 10, about 99%, or about 10, about 99%, or about 10, about 99, about 10, about 99, about, or about 99, about 99, about, or about, about.

In some cases, the devices, systems, kits, and methods disclosed herein require at least about 100 μ L saliva to provide a test result with at least about 95% confidence or accuracy.

In some cases, the devices, systems, kits, and methods disclosed herein require at least about 50 μ L vaginal fluid to provide test results with at least about 95% confidence or accuracy.in some cases, the devices, systems, kits, and methods disclosed herein require at least about 100 μ L vaginal fluid to provide test results with at least about 95% confidence or accuracy.in some cases, the devices, systems, kits, and methods disclosed herein require at least about 200 μ L vaginal fluid to provide test results with at least about 95% confidence or accuracy.in some cases, the devices, systems, kits, and methods disclosed herein require at least about 500 μ L vaginal fluid to provide test results with at least about 95% confidence or accuracy.in some cases, the devices, systems, kits, and methods disclosed herein require at least about 1ml vaginal fluid to provide test results with at least about 95% confidence or accuracy.in some cases, the devices, systems, kits, and methods disclosed herein require at least about 2ml vaginal fluid to provide test results with at least about 95% confidence or accuracy.

Cell-free nucleic acids

In some cases, the methods, devices, systems, and kits disclosed herein can be used to assess cell-free nucleic acids in a biological sample. In some cases, the cell-free nucleic acid is DNA (cf-DNA) or RNA (cf-RNA). In some cases, the cell-free nucleic acid is fetal nucleic acid. In some cases, the cell-free fetal nucleic acid is cell-free fetal DNA (cff-DNA) or cell-free fetal RNA (cff-RNA). In some cases, the cf-DNA or cff-DNA is genomic DNA or cDNA. In some cases, the cf-DNA comprises mitochondrial DNA. In some cases, cf-RNA or cff-RNA is messenger RNA (mrna), microrna (mirna), mitochondrial RNA, or natural antisense RNA (NAS-RNA). In some cases, the cell-free nucleic acid is a mixture of maternal and fetal nucleic acids. Circulating cell-free fetal nucleic acid in the maternal blood stream may be referred to as "circulating cell-free nucleic acid" or "circulating extracellular DNA". In some cases, the cell-free nucleic acid comprises an epigenetic modification. In some cases, the cell-free nucleic acid comprises a pattern of epigenetic modifications that correspond to gender or other genetic information of interest. In some cases, the cell-free nucleic acid comprises a methylated cytosine. In some cases, the cell-free nucleic acid comprises a cytosine methylation pattern that corresponds to gender or other genetic information of interest.

In some cases, the methods, devices, systems, and kits disclosed herein are configured to detect or quantify cellular nucleic acids, such as nucleic acids from a disrupted or lysed cell. In some cases, the cellular nucleic acid is from a cell that is intentionally disrupted or lysed. In some cases, the cellular nucleic acid is from a cell that is unintentionally disrupted or lysed. The methods, devices, systems, and kits disclosed herein can be configured to analyze cells that are intentionally destroyed or lysed, rather than cells that are unintentionally destroyed or lysed. In some cases, less than about 0.1% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 1% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 5% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 10% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 20% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 30% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 40% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 50% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 60% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 70% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 80% of the total nucleic acids in the biological sample are cellular nucleic acids. In some cases, less than about 90% of the total nucleic acids in the biological sample are cellular nucleic acids.

Experimental control

In some cases, the devices, systems, kits, and methods comprise experimental controls or uses thereof. In some cases, experimental controls include nucleic acids, proteins, peptides, antibodies, antigen-binding antibody fragments, binding moieties. In some cases, the experimental control comprises a signal for detecting the experimental control. Non-limiting examples of signals are fluorescent molecules, dye molecules, nanoparticles, and colorimetric indicators. In some cases, the experimental control comprises cell-free nucleic acid. In some cases, the cell-free nucleic acid comprises cell-free fetal nucleic acid. In some cases, the cell-free nucleic acid comprises a maternal cell-free nucleic acid. In some cases, the cell-free nucleic acid includes maternal cell-free nucleic acid (e.g., to assess the amount of cell destruction/lysis that occurs during sample processing). In some cases, the cell-free nucleic acid comprises a sequence corresponding to the Y chromosome. In some cases, the cell-free nucleic acid comprises a sequence corresponding to the X chromosome. In some cases, the cell-free nucleic acid comprises a sequence corresponding to an autosome. In some cases, the experimental control is a fetal nucleic acid control. In some cases, there are regions of DNA that are differentially methylated, which indicates the presence of fetal DNA. In some cases, the fetal DNA control provides confirmation of pregnancy. By way of non-limiting example, the RASSF1A gene has been reported to be hypermethylated in placental cells and hypomethylated in maternal blood cells.

In some cases, the biological sample is a maternal bodily fluid sample obtained from a pregnant subject, a suspected pregnant subject, or a subject that has recently (e.g., within the past day) born. In some cases, the maternal body fluid sample comprises blood, such as whole blood, a peripheral blood sample or a blood fraction (plasma, serum). In some cases, the maternal body fluid sample comprises sweat, tears, sputum, urine, ear fluid, lymph fluid, saliva, cerebrospinal fluid, bone marrow suspensions, vaginal fluid, transcervical lavage fluid, brain fluid, ascites, milk, respiratory, intestinal and genitourinary secretions, amniotic fluid or leukapheresis (leukaphoresis) samples. In some cases, the biological sample is a maternal bodily fluid sample that can be readily obtained by non-invasive surgery, such as blood, plasma, serum, sweat, tears, sputum, urine, ear fluid, or saliva. In some cases, the sample is a combination of at least two bodily fluid samples. In some cases, the cell-free fetal nucleic acid is derived from a maternal placenta, e.g., from apoptotic placental cells. In some cases, the biological sample is placental blood.

In some cases, the nucleic acids evaluated or analyzed by the devices, systems, kits, and methods disclosed herein are of a preferred length. In some cases, the nucleic acid is a cell-free fetal DNA fragment. In some cases, the cell-free fetal DNA fragment is from the Y chromosome. In some cases, the nucleic acid is about 15bp to about 500bp in length. In some cases, the nucleic acid is about 50bp to about 200bp in length. In some cases, the nucleic acid is at least about 15bp in length. In some cases, the nucleic acid is at most about 500bp in length. In some cases, the nucleic acid has a length of about 15bp to about 50bp, about 15bp to about 75bp, about 15bp to about 100bp, about 15bp to about 150bp, about 15bp to about 200bp, about 15bp to about 250bp, about 15bp to about 300bp, about 15bp to about 350bp, about 15bp to about 400bp, about 15bp to about 450bp, about 15bp to about 500bp, about 50bp to about 75bp, about 50bp to about 100bp, about 50bp to about 150bp, about 50bp to about 200bp, about 50bp to about 250bp, about 50bp to about 300bp, about 50bp to about 350bp, about 50bp to about 400bp, about 50bp to about 450bp, about 50bp to about 500bp, about 75bp to about 100bp, about 75bp to about 150bp, about 75bp to about 200bp, about 75bp to about 250bp, about 75bp to about 300bp, about 50bp to about 75bp, about 350bp to about 75bp, about 75bp to about 500bp, about 75bp, about 50bp to about 500bp, about 50bp, about 75bp, about 50bp, about 100bp to about 200bp, about 100bp to about 250bp, about 100bp to about 300bp, about 100bp to about 350bp, about 100bp to about 400bp, about 100bp to about 450bp, about 100bp to about 500bp, about 150bp to about 200bp, about 150bp to about 250bp, about 150bp to about 300bp, about 150bp to about 350bp, about 150bp to about 400bp, about 150bp to about 450bp, about 150bp to about 500bp, about 200bp to about 250bp, about 200bp to about 300bp, about 200bp to about 350bp, about 200bp to about 400bp, about 200bp to about 450bp, about 200bp to about 500bp, about 250bp to about 300bp, about 250bp to about 350bp, about 250bp to about 400bp, about 250bp to about 450bp, about 300bp to about 350bp, about 300bp to about 350bp, about 350bp to about 450bp, about 400bp to about 500bp, about 300bp to about 450bp, about 350bp to about 350bp, about 500bp to about 500bp, about 300bp to about 450bp, about 500bp to about 500bp, about 500bp to about 500bp, From about 400bp to about 500bp or from about 450bp to about 500 bp. In some cases, the nucleic acid is about 15bp, about 50bp, about 75bp, about 100bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, or about 500bp in length.

The size of the cell-free nucleic acid assessed using the methods, devices, systems, and kits disclosed herein can vary depending, for example, on the particular bodily fluid sample used. For example, it has been observed that cff-DNA sequence is shorter than the parent cf-DNA sequence, and that both cff-DNA and the parent cf-DNA in urine are shorter than in plasma samples.

In some cases, the length of the cff-DNA sequence evaluated in urine is from about 20bp to about 300 bp. In some cases, the length of the cff-DNA sequence evaluated in the urine sample is from about 15bp to about 300 bp. In some cases, the length of the cff-DNA sequence evaluated in the urine sample is at least about 15 bp. In some cases, the length of the cff-DNA sequence evaluated in the urine sample is at most about 300 bp. In some cases, the length of the cff-DNA sequence evaluated in the urine sample is from about 15bp to about 20bp, from about 15bp to about 30bp, from about 15bp to about 60bp, from about 15bp to about 90bp, from about 15bp to about 120bp, from about 15bp to about 150bp, from about 15bp to about 180bp, from about 15bp to about 210bp, from about 15bp to about 240bp, from about 15bp to about 270bp, from about 15bp to about 300bp, from about 20bp to about 30bp, from about 20bp to about 60bp, from about 20bp to about 90bp, from about 20bp to about 120bp, from about 20bp to about 150bp, from about 20bp to about 180bp, from about 20bp to about 210bp, from about 20bp to about 240bp, from about 20bp to about 270bp, from about 20bp to about 300bp, from about 30bp to about 60bp, from about 30bp to about 90bp, from about 30bp to about 120bp, from about 30bp to about 150bp, from about 30bp to about 180bp, from about 30bp to about 240bp, from about 30bp to about 180bp, from about 30bp, from about 180bp, from about 120bp, from about, About 30bp to about 300bp, about 60bp to about 90bp, about 60bp to about 120bp, about 60bp to about 150bp, about 60bp to about 180bp, about 60bp to about 210bp, about 60bp to about 240bp, about 60bp to about 270bp, about 60bp to about 300bp, about 90bp to about 120bp, about 90bp to about 150bp, about 90bp to about 180bp, about 90bp to about 210bp, about 90bp to about 240bp, about 90bp to about 270bp, about 90bp to about 300bp, about 120bp to about 150bp, about 120bp to about 180bp, about 120bp to about 210bp, about 120bp to about 240bp, about 120bp to about 270bp, about 120bp to about 300bp, about 150bp to about 180bp, about 150bp to about 210bp, about 150bp to about 240bp, about 150bp to about 270bp, about 180bp to about 180bp, about 180bp to about 180bp, about 180bp, About 210bp to about 300bp, about 240bp to about 270bp, about 240bp to about 300bp, or about 270bp to about 300 bp. In some cases, the length of the cff-DNA sequence evaluated in the urine sample is about 15bp, about 20bp, about 30bp, about 60bp, about 90bp, about 120bp, about 150bp, about 180bp, about 210bp, about 240bp, about 270bp, or about 300 bp.

In some cases, the length of the cff-DNA sequence evaluated in the plasma or serum sample is at least about 20 bp. In some cases, the length of the cff-DNA sequence evaluated in the plasma or serum sample is at least about 40 bp. In some cases, the length of the cff-DNA sequence evaluated in the plasma or serum sample is at least about 80 bp. In some cases, the length of the cff-DNA sequence evaluated in the plasma or serum sample is at most about 500 bp. In some cases, the length of the cff-DNA sequence evaluated in plasma or serum is from about 100bp to about 500 bp. In some cases, the length of the cff-DNA sequence evaluated in the plasma or serum sample is from about 50bp to about 500 bp. In some cases, the cff-DNA sequence evaluated in the plasma or serum sample has a length of about 80bp to about 100bp, about 80bp to about 125bp, about 80bp to about 150bp, about 80bp to about 175bp, about 80bp to about 200bp, about 80bp to about 250bp, about 80bp to about 300bp, about 80bp to about 350bp, about 80bp to about 400bp, about 80bp to about 450bp, about 80bp to about 500bp, about 100bp to about 125bp, about 100bp to about 150bp, about 100bp to about 175bp, about 100bp to about 200bp, about 100bp to about 250bp, about 100bp to about 300bp, about 100bp to about 350bp, about 100bp to about 400bp, about 100bp to about 450bp, about 100bp to about 500bp, about 125bp to about 150bp, about 125bp to about 125bp, about 125bp to about 300bp, about 125bp to about 200bp, about 125bp to about 125bp, about 125bp to about 450bp, about 100bp to about 125bp to about 300bp, about 125bp to about 300, About 125bp to about 500bp, about 150bp to about 175bp, about 150bp to about 200bp, about 150bp to about 250bp, about 150bp to about 300bp, about 150bp to about 350bp, about 150bp to about 400bp, about 150bp to about 450bp, about 150bp to about 500bp, about 175bp to about 200bp, about 175bp to about 250bp, about 175bp to about 300bp, about 175bp to about 350bp, about 175bp to about 400bp, about 175bp to about 450bp, about 175bp to about 500bp, about 200bp to about 250bp, about 200bp to about 300bp, about 200bp to about 350bp, about 200bp to about 400bp, about 200bp to about 450bp, about 200bp to about 500bp, about 250bp to about 300bp, about 250bp to about 350bp, about 250bp to about 400bp, about 250bp to about 450bp, about 250bp to about 300bp, about 300bp to about 350bp, about 350bp to about 350bp, about 300bp to about 300bp, about 300bp, From about 350bp to about 500bp, from about 400bp to about 450bp, from about 400bp to about 500bp, or from about 450bp to about 500 bp. In some cases, the estimated length of the cff-DNA sequence in the plasma or serum sample is about 80bp, about 100bp, about 125bp, about 150bp, about 175bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, or about 500 bp.

In some cases, unless otherwise stated, cell-free nucleic acid comprises sequences present on the human Y chromosome, referred to herein as "Y chromosome sequences". in some cases, cell-free nucleic acid comprises sequences found only on the Y chromosome. in some cases, cell-free nucleic acid comprises sequences not found on the X chromosome or any autosome.in some cases, at least a portion of a Y chromosome sequence is found in the Y chromosome protein-encoding gene.

Test subject

Disclosed herein are devices, systems, kits, and methods for analyzing biological components in a sample from a subject. The subject may be a human. The subject may be a non-human. The subject may be a non-mammal (e.g., bird, reptile, insect). In some cases, the subject is a mammal. In some cases, the mammal is a female. In some cases, the subject is a human subject. In some cases, the mammal is a primate (e.g., human, great ape, lesser ape, monkey). In some cases, the mammal is a canine (e.g., a dog, a fox, a wolf). In some cases, the mammal is a feline (e.g., domestic cat, large feline). In some cases, the mammal is an equine (e.g., horse). In some cases, the mammal is a bovine (e.g., cow, buffalo, bison). In some cases, the mammal is a sheep. In some cases, the mammal is a goat. In some cases, the mammal is a pig. In some cases, the mammal is a rodent (e.g., mouse, rat, rabbit, guinea pig).

In some cases, a subject described herein is affected by a disease or condition. The devices, systems, kits, and methods disclosed herein can be used to test for, detect, and/or monitor a disease or condition. The devices, systems, kits, and methods disclosed herein can be used to test for the presence of genetic traits, monitor fitness, and determine family connections.

The devices, systems, kits, and methods disclosed herein can be used to test, detect, and/or monitor cancer in a subject. Non-limiting examples of cancer include breast cancer, prostate cancer, skin cancer, lung cancer, colorectal/colon cancer, bladder cancer, pancreatic cancer, lymphoma, and leukemia.

The devices, systems, kits, and methods disclosed herein can be used to test, detect, and/or monitor an immune disorder or an autoimmune disorder in a subject. Autoimmune and immune disorders include, but are not limited to, type 1 diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, lupus, inflammatory bowel disease, Addison's disease, Graves ' disease, Crohn's disease, and celiac disease.

The devices, systems, kits, and methods disclosed herein can be used to test, detect, and/or monitor a disease or condition associated with aging in a subject. Diseases and conditions associated with aging include, but are not limited to, cancer, osteoporosis, dementia, macular degeneration, metabolic conditions, and neurodegenerative disorders.

For example, detecting thrombophilia may include detecting a polymorphism present in a gene selected from the group consisting of factor V L eiden (FV L), the prothrombin gene (PT G20210A), and methylenetetrahydrofolate reductase (MTHFR).

Non-limiting examples of neurodegenerative and neurological disorders are alzheimer ' S disease, parkinson ' S disease, huntington ' S disease, spinocerebellar ataxia, amyotrophic lateral sclerosis (a L S), motor neuron disease, chronic pain, and spinal muscular atrophy.

The devices, systems, kits, and methods disclosed herein can be used to test, detect, and/or monitor metabolic conditions or diseases. Metabolic conditions and diseases include, but are not limited to, obesity, thyroid disorders, hypertension, type 1 diabetes, type 2 diabetes, nonalcoholic steatohepatitis, coronary artery disease, and atherosclerosis.

The devices, systems, kits, and methods disclosed herein may be used to test, detect, and/or monitor allergy or intolerance to food, liquids, or drugs. By way of non-limiting example, the subject may be allergic or intolerant to lactose, wheat, soy, dairy products, caffeine, alcohol, nuts, shellfish, and eggs. The subject may also be allergic or intolerant to drugs, supplements or cosmetics. In some cases, the method includes analyzing a genetic marker predictive of skin type or skin health.

In some cases, the condition is associated with allergy. In some cases, the subject is not diagnosed with a disease or condition, but is experiencing symptoms indicative of the presence of a disease or condition. In other cases, the subject has been diagnosed with a disease or condition, and the devices, systems, kits, and methods disclosed herein can be used to monitor the disease or condition, or the effect of a drug on the disease or condition.

The devices, systems, kits, and methods disclosed herein may be used to test, detect, and/or monitor pregnancy. In some cases, the subject is a pregnant subject in the first, middle or last trimester of pregnancy. In some cases, the pregnant subject is at less than about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27 weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39 weeks, or about 40 weeks of gestation.

In some cases, the pregnant subject is about 2 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 3 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 4 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 5 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 6 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 7 weeks gestation to about 42 weeks gestation. In some cases, the pregnant subject is about 8 weeks gestation to about 42 weeks gestation.

In some cases, the pregnant subject has reached at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, or at least about 8 weeks of gestation. In some cases, the pregnant subject has reached at least about 5 weeks to about 8 weeks of gestation. In some cases, the pregnant subject has reached a pregnancy of at least about 5 weeks to about 8 weeks, at least about 5 weeks to about 12 weeks, at least about 5 weeks to about 16 weeks, at least about 5 weeks to about 20 weeks, at least about 6 weeks to about 21 weeks, at least about 6 weeks to about 22 weeks, at least about 6 weeks to about 24 weeks, at least about 6 weeks to about 26 weeks, at least about 6 weeks to about 28 weeks, at least about 6 weeks to about 9 weeks, at least about 6 weeks to about 12 weeks, at least about 6 weeks to about 16 weeks, at least about 6 weeks to about 20 weeks, at least about 6 weeks to about 21 weeks, at least about 6 weeks to about 22 weeks, at least about 6 weeks to about 24 weeks, at least about 6 weeks to about 26 weeks, or at least about 6 weeks to about 28 weeks. In some cases, the pregnant subject has reached a pregnancy of at least about 7 weeks to about 8 weeks, at least about 7 weeks to about 12 weeks, at least about 7 weeks to about 16 weeks, at least about 7 weeks to about 20 weeks, at least about 7 weeks to about 21 weeks, at least about 7 weeks to about 22 weeks, at least about 7 weeks to about 24 weeks, at least about 7 weeks to about 26 weeks, at least about 7 weeks to about 28 weeks, at least about 8 weeks to about 9 weeks, at least about 8 weeks to about 12 weeks, at least about 6 weeks to about 16 weeks, at least about 8 weeks to about 20 weeks, at least about 8 weeks to about 21 weeks, at least about 6 weeks to about 22 weeks, at least about 8 weeks to about 24 weeks, at least about 8 weeks to about 26 weeks, or at least about 8 weeks to about 28 weeks. In some cases, the gestational period is determined by starting the measurement from the first day of the last menstrual period.

The devices, systems, kits, and methods disclosed herein are not limited to medical or health-related applications. For example, the devices, systems, kits, and methods disclosed herein may be used in the field of forensics or to detect blood transfusion (blood transfusion).

Numbering embodiments

The disclosure is further understood by reviewing the numbered embodiments listed herein. 1. An apparatus, comprising: a sample purifier for removing cells from a biological fluid sample to produce a cell-removed sample; at least one of a detection reagent and a signal detector to detect a plurality of biomarkers in the cell removal sample. 2. The apparatus of embodiment 1, wherein the plurality of biomarkers comprises a plurality of cell-free DNA fragments. 3. The apparatus of embodiment 2, wherein each of the plurality of cell-free fragments comprises a region represented by a first sequence or a second sequence that is at least 90% homologous to the first sequence. 4. The apparatus of embodiment 1, wherein the plurality of biomarkers are nucleic acids, and wherein the apparatus comprises at least one nucleic acid amplification reagent and at least one oligonucleotide having a sequence corresponding to a target nucleic acid. 5. The apparatus of embodiment 4, wherein the at least one nucleic acid amplification reagent comprises an oligonucleotide primer capable of amplifying a region of a chromosome having a first sequence that is similar to a second sequence in a genome of a subject, and wherein the first sequence is sufficiently physically distant from the second sequence such that the first sequence is present on a first cell-free nucleic acid of the subject and the second sequence is present on a second cell-free nucleic acid of the subject. 6. The apparatus of embodiment 5, wherein at least one of the first sequence and the second sequence is repeated at least five times in the genome of the subject. 7. The apparatus of embodiment 5, wherein the first sequence and the second sequence are each at least 10 nucleotides in length. 8. The apparatus of embodiment 5, wherein the first sequence is on a first chromosome and the second sequence is on a second chromosome. 9. The device of embodiment 5, wherein the first sequence and the second sequence are on the same chromosome, but separated by at least 1 nucleotide. 10. The apparatus of embodiment 5, wherein the first sequence and the second sequence are functionally linked. 11. The apparatus of embodiment 5, wherein the first sequence is at least 80% identical to the second sequence. 12. The device of embodiment 1, wherein the biomarker is a cell-free nucleic acid. 13. The device of embodiment 1, wherein the aggregates contain at least two biomarkers. 14. The apparatus of embodiment 1, wherein the sample purifier comprises a filter. 15. The apparatus of embodiment 14, wherein the sample purifier comprises a wicking material or capillary device for pushing the biological fluid through the filter. 16. The apparatus of embodiment 14, wherein the filter has a pore size of about 0.05 microns to about 2 microns. 17. The apparatus of embodiment 1, wherein the sample purifier comprises a binding moiety that binds to a nucleic acid, a protein, a cell surface marker, or a microbubble surface marker in the fluid sample. 18. The device of embodiment 17, wherein the binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof. 19. The apparatus of embodiment 17, wherein said binding moiety is capable of binding to an extracellular vesicle, wherein said extracellular vesicle is released from a fetal cell or a placental cell of said female subject. 20. The apparatus of embodiment 4, wherein the at least one nucleic acid amplification reagent comprises at least one isothermal amplification reagent. 21. The apparatus of embodiment 20, wherein the at least one isothermal amplification reagent comprises a recombinase polymerase, a single-stranded DNA binding protein, a strand-displacing polymerase, or a combination thereof. 22. The apparatus of embodiment 1, wherein the signal detector comprises a solid support. 23. The apparatus of embodiment 22, wherein the solid support is a column. 24. The apparatus of embodiment 22, wherein the solid support comprises a binding moiety that binds to the amplification product. 25. The apparatus of embodiment 24, wherein the binding moiety is an oligonucleotide. 26. The device of embodiment 1, wherein the signal detector is a lateral flow strip. 27. The apparatus of embodiment 26, wherein the detection reagent comprises gold particles or fluorescent particles. 28. The apparatus of embodiment 1, the sample purifier removes cells from blood, and the cell-removed sample is plasma. 29. The apparatus of embodiment 1, wherein the apparatus is contained in a single housing. 30. The apparatus of embodiment 1, wherein the apparatus operates at room temperature. 31. The apparatus of embodiment 4, wherein the apparatus detects the amplification product within about five minutes to about twenty minutes of receiving the biological fluid. 32. The apparatus of embodiment 1, comprising a transport or storage compartment. 33. The apparatus of embodiment 32, wherein the transport or storage compartment comprises an absorbent pad or a fluid container. 34. The apparatus of embodiment 1, comprising a communication connection. 35. The apparatus of embodiment 34, wherein the communication connection is a wireless communication system, a cable, or a cable port. 36. The apparatus of embodiment 1, comprising a percutaneous puncture device. 37. A method, comprising: obtaining a fluid sample from a subject, wherein the volume of the biological sample is no greater than about 300 μ Ι _; contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sequence of interest; and detecting the presence or absence of amplification products, wherein the presence or absence is indicative of the health state of the subject. 38. The method of embodiment 37, wherein the fluid sample is a blood sample. 39. The method of embodiment 38, wherein the volume of the blood sample is no greater than 120 μ Ι. 40. The method of embodiment 37, wherein the fluid sample is a plasma sample from blood. 41. The method of embodiment 40, wherein said volume of said plasma sample is no greater than 50 μ l. 42. The method of embodiment 40, wherein said volume of said plasma sample is from about 10 μ l to about 40 μ l. 43. The method of any one of embodiments 37-42, wherein obtaining comprises performing a finger prick. 44. The method of embodiment 43 comprising squeezing a pricked finger to increase blood from the fingerstick. 45. The method of embodiment 38, wherein obtaining the blood sample does not comprise performing a phlebotomy. 46. The method of embodiment 37, wherein the fluid sample is a urine sample. 47. The method of embodiment 37, wherein the fluid sample is a saliva sample. 48. The method of any one of embodiments 37-47, comprising removing at least one of cells, cell fragments, and microparticles from the fluid sample. 49. The method of embodiment 37, wherein the sample contains about 25pg to about 250pg of total circulating cell-free DNA. 50. The method of embodiment 49, wherein the sample comprises cell-free DNA fragments having a length of from about 20 base pairs to about 160 base pairs. 51. The method of embodiment 37, wherein the sample contains about 5 to about 100 copies of a sequence of interest. 52. The method of embodiment 51, wherein the sequence of interest is at least 10 nucleotides in length. 53. The method of embodiment 51, wherein said 100 copies are at least 90% identical to each other. 54. The method of embodiment 37, wherein amplifying comprises isothermal amplification. 55. The method of embodiment 37, wherein amplification occurs at room temperature. 56. The method of embodiment 37, wherein the method comprises incorporating a tag into the amplification products while the amplification occurs, and wherein detecting the at least one amplification product comprises detecting the tag. 57. The method of embodiment 56, wherein said tag does not comprise a nucleotide. 58. The method of embodiment 57, wherein detecting the amplification product comprises contacting the amplification product with a binding moiety capable of interacting with the tag. 59. The method of embodiment 58, comprising contacting the amplification product with the binding moiety on a lateral flow device. 60. The method of embodiment 37, wherein steps (a) through (c) are performed in less than fifteen minutes. 61. The method of embodiment 37, wherein the method is performed by the subject. 62. The method of embodiment 37, wherein the method is performed by an individual who has not received technical training for performing the method. 63. The method of embodiment 37, comprising obtaining, contacting and detecting with a single handheld device. 64. The method of embodiment 63, wherein said obtaining is performed by said subject pressing their skin against a transcutaneous puncturing device of a handheld device. 65. The method of embodiment 64, wherein said subject presses his skin against said percutaneous puncture device no more than once. 66. The method of embodiment 64, wherein said subject presses his skin against said transcutaneous puncture device no more than twice. 67. The method of embodiment 37, wherein said health state is selected from the presence or absence of pregnancy. 68. The method of embodiment 37, wherein the health state is selected from the group consisting of the presence or absence of a neurological disorder, a metabolic disorder, cancer, an autoimmune disorder, allergy, and infection. 69. The method of embodiment 37, wherein the health state is a response to a drug or treatment. 70. An apparatus, comprising: a sample purifier that removes cells from a fluid sample of a female subject; at least one nucleic acid amplification reagent; at least one oligonucleotide comprising a sequence corresponding to the Y chromosome, wherein the at least one oligonucleotide and nucleic acid amplification reagents are capable of producing an amplification product; and at least one of a detection reagent or a signal detector for detecting the amplification product. 71. The apparatus of embodiment 70, wherein the fluid sample is blood. 72. The apparatus of embodiment 70, wherein said sample purifier comprises a filter. 73. The apparatus of embodiment 72, wherein the sample purifier comprises a wicking material or capillary device to push the biological fluid through the filter. 74. The apparatus of embodiment 72, wherein the filter has a pore size of about 0.05 microns to about 2 microns. 75. The apparatus of embodiment 70, wherein the sample purifier comprises a binding moiety that binds to a nucleic acid, a protein, a cell surface marker, or a microbubble surface marker in the fluid sample. 76. The device of embodiment 75, wherein said binding moiety comprises an antibody, an antigen-binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof. 77. The apparatus of embodiment 76, wherein said binding moiety is capable of binding to an extracellular vesicle, wherein said extracellular vesicle is released from a fetal cell or a placental cell of said female subject. 78. The apparatus of embodiment 76, wherein said binding moiety binds to human chorionic gonadotropin protein or a transcript of a human chorionic gonadotropin encoding gene. 79. The apparatus of embodiment 70, wherein the at least one oligonucleotide comprises a primer that hybridizes to a Y chromosome sequence. 80. The apparatus of embodiment 70, wherein the at least one oligonucleotide comprises a probe that hybridizes to a nucleic acid represented by a Y chromosome sequence or a transcript thereof, and wherein the probe comprises an oligonucleotide tag. 81. The apparatus of embodiment 80, wherein the oligonucleotide tag is not specific for a Y chromosome sequence. 82. The apparatus of embodiment 80 or 81, wherein said apparatus comprises at least one primer that hybridizes to said oligonucleotide tag and produces an amplification product in the presence of said amplification reagents. 83. The apparatus of embodiment 80, wherein the Y chromosome sequence is a sequence located between position 20082183 and position 20350897 of the Y chromosome. 84. The apparatus of embodiment 80, wherein the Y chromosome sequence is a sequence located between position 20350799 and position 20350897 of the Y chromosome. 85. The apparatus of embodiment 80, wherein the Y chromosome sequence is a sequence located between position 20349236 and position 20349318 of the Y chromosome. 86. The apparatus of embodiment 80, wherein the Y chromosome sequence is a sequence located between position 20082183 and position 20350897 of the Y chromosome. 87. The apparatus of embodiment 80, wherein the Y chromosome sequence is a sequence located between position 20350601 and position 20350699 of the Y chromosome. 88. The apparatus of embodiment 80, wherein said Y chromosome sequence is a sequence located between position 20082183 and position 20082281 of the Y chromosome. 89. The device of embodiment 80, wherein the Y chromosome sequence is a sequence located in a gene selected from the group consisting of DYS14 gene or TTTY 22. 90. The apparatus of any one of embodiments 83-89, wherein the sequence is at least about 10 nucleotides in length. 91. The apparatus of embodiment 70, wherein the at least one nucleic acid amplification reagent comprises at least one isothermal amplification reagent. 92. The apparatus of embodiment 83, wherein the at least one isothermal amplification reagent comprises a recombinase polymerase, a single-stranded DNA binding protein, a strand-displacing polymerase, or a combination thereof. 93. The apparatus of embodiment 70, wherein the signal detector comprises a solid support. 94. The apparatus of embodiment 93, wherein the solid support is a column. 95. The apparatus of embodiment 93, wherein the solid support comprises a binding moiety that binds to the amplification product. 96. The apparatus of embodiment 95, wherein said binding moiety is an oligonucleotide. 97. The device of embodiment 70, wherein the signal detector is a lateral flow strip. 98. The apparatus of embodiment 97, wherein the detection reagent comprises gold particles. 99. The apparatus of embodiment 97, wherein the detection reagent comprises a fluorescent particle. 100. The apparatus of embodiment 70, wherein the apparatus is contained in a single housing. 101. The apparatus of embodiment 70, wherein the apparatus operates at room temperature. 102. The apparatus of embodiment 70, wherein said apparatus detects said amplification product within about five minutes to about twenty minutes of receiving said biological fluid. 103. The apparatus of embodiment 70, comprising a transport or storage compartment. 104. The apparatus of embodiment 103, wherein the transport or storage compartment comprises an absorbent pad or a fluid container. 105. The apparatus of implementation 70, comprising a communication connection. 106. The apparatus of embodiment 105, wherein said communication connection is a wireless communication system, a cable, or a cable port. 107. The apparatus of embodiment 70, comprising a percutaneous puncture device. 108. A kit comprising the device according to any one of embodiments 70-107, and a component selected from the group consisting of a structure or a reagent for obtaining a sample, purifying an analyte in the sample, amplifying the analyte, and detecting the analyte. 109. The kit of embodiment 108, wherein the component for obtaining a sample is a transcutaneous puncture device. 110. The kit of embodiment 109, comprising a capillary tube for drawing blood from a percutaneous puncture. 111. The kit of embodiment 108, comprising a container, pouch, wire or cable for heating or cooling the device's components thereof. 112. A method, comprising: obtaining a fluid sample from a female pregnant subject, wherein the volume of the biological sample is no greater than about 300 μ Ι _; contacting at least one cell-free nucleic acid in the fluid sample with an amplification reagent and an oligonucleotide primer that anneals to a sequence corresponding to a sex chromosome; and detecting the presence or absence of amplification product, wherein the presence or absence is indicative of the sex of the fetus of the female pregnant subject. 113. The method of embodiment 112, wherein the fluid sample is a blood sample. 114. The method of embodiment 113, wherein the volume of said blood sample is no greater than 120 μ Ι. 115. The method of embodiment 112, wherein the fluid sample is a plasma sample from blood. 116. The method according to embodiment 115, wherein the volume of the plasma sample is no greater than 50 μ Ι. 117. The method according to embodiment 115, wherein the volume of the plasma sample is from about 10 μ Ι to about 40 μ Ι. 118. The method according to any one of embodiments 112-117, wherein obtaining comprises performing a finger prick. 119. The method of embodiment 118, comprising squeezing the pricked finger to increase blood from the fingerstick. 120. The method of embodiment 113, wherein obtaining the blood sample does not comprise performing a phlebotomy. 121. The method of embodiment 112, wherein the fluid sample is a urine sample. 122. The method of embodiment 112, wherein the fluid sample is a saliva sample. 123. The method of any one of embodiments 112-122, comprising removing at least one of cells, cell fragments, and microparticles from the fluid sample. 124. The method of embodiment 112, wherein the sample contains about 25pg to about 250pg of total circulating cell-free DNA. 125. The method of embodiment 112, wherein the cell-free nucleic acid comprises cell-free fetal DNA fragments. 126. The method of embodiment 125, wherein the cell-free fetal DNA fragments are about 20 base pairs to about 160 base pairs in length. 127. The method of embodiment 112, wherein the sequence corresponding to the sex chromosome is a Y chromosome sequence that is present on the Y chromosome in at least two copies. 128. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20082183 and position 20350897 of the Y chromosome. 129. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20350799 and position 20350897 of the Y chromosome. 130. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20349236 and position 20349318 of the Y chromosome. 131. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20082183 and position 20350897 of the Y chromosome. 132. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20350601 and position 20350699 of the Y chromosome. 133. The method of embodiment 127, wherein the Y chromosome sequence is a sequence located between position 20082183 and position 20082281 of the Y chromosome. 134. The method of embodiment 127, wherein the Y chromosome sequence is a sequence found in the DYS14 gene or TTTY22 gene. 135. The method of any one of embodiments 127-134, wherein the sequence is at least about 10 nucleotides in length. 136. The method of embodiment 112, wherein the sample does not contain more than about 100 copies of the cell-free nucleic acid. 137. The method of embodiment 112, wherein the sample contains about 5 to about 100 copies of the cell-free nucleic acid. 138. The method of embodiment 112, wherein said female pregnant subject is no more than 8 weeks pregnant. 139. The method of embodiment 112, wherein amplifying comprises isothermal amplification. 140. The method of embodiment 112, wherein amplification occurs at room temperature. 141. The method of embodiment 112, wherein amplifying comprises contacting the circulating cell-free nucleic acid with a recombinase polymerase. 142. The method of embodiment 112, comprising labeling the cell-free nucleic acid with an oligonucleotide tag. 143. The method of embodiment 112, wherein amplifying comprises contacting the cell-free nucleic acid with at least one oligonucleotide primer having a sequence corresponding to the oligonucleotide tag. 144. The method of embodiment 143, wherein the oligonucleotide primer comprises a blocking group that prevents extension of the oligonucleotide primer until at least one of amplification conditions and amplification reagents are provided. 145. The method of embodiment 112, wherein the method comprises incorporating a tag into the amplification products while the amplification occurs, and wherein detecting the at least one amplification product comprises detecting the tag. 146. The method of embodiment 145, wherein detecting the amplification product comprises detecting an amplified oligonucleotide tag. 147. The method of embodiment 145, wherein the tag comprises a nucleotide. 148. The method of embodiment 145, wherein the tag does not comprise a nucleotide. 149. The method of embodiment 145, wherein detecting the amplification product comprises contacting the amplification product with a binding moiety capable of interacting with the tag or oligonucleotide tag. 150. The method of embodiment 149, comprising contacting the amplification product with the binding moiety on a lateral flow device. 151. The method of embodiment 112, wherein steps (a) through (c) are performed in less than fifteen minutes. 152. The method of embodiment 112, wherein the method is performed by the subject. 153. The method of embodiment 112, wherein the method is performed by an individual who has not received technical training for performing the method. 154. A method, comprising: obtaining a fluid sample from a female pregnant subject with a handheld device, wherein the fluid sample has a volume of no greater than about 300 μ Ι _; sequencing at least one cell-free nucleic acid in the fluid sample with the handheld device; detecting, by a display in the handheld device, the presence or absence of a sequence corresponding to the Y chromosome, thereby determining the gender of the fetus in the female pregnant subject; and communicating the gender to another subject with the handheld device. 155. The method of embodiment 154, wherein detecting and delivering occur simultaneously. 156. The method of embodiment 154, wherein the volume is no greater than 120 μ Ι _. 157. The method of embodiment 154, wherein obtaining does not comprise phlebotomy. 158. The method of embodiment 154, wherein said female pregnant subject performs said obtaining by pressing their skin against a transcutaneous penetration device of said handheld apparatus. 159. The method of embodiment 158, wherein said female pregnant subject has a finger pressed against said percutaneous puncture device. 160. The method of embodiment 158, wherein said female pregnant subject presses their skin against said percutaneous puncture device no more than once. 161. The method of embodiment 158, wherein said female pregnant subject presses their skin against said percutaneous puncture device no more than twice.