阅读说明：本技术 文库定量和鉴定 (Library quantification and identification ) 是由 D·A·阿莫雷塞 B·李 B·G·施罗德 R·A·费克特 于 2017-04-11 设计创作，主要内容包括：本文描述了用于文库定量和鉴定的方法、组合物和试剂盒。一些实施例涉及一种文库定量的方法。例如,所述方法可以包含提供DNA片段,在存在各自标记有荧光团的引物的情况下通过聚合酶链反应PCR扩增所述DNA片段。在这些情况下,仅预定数量的荧光团连接到每个DNA片段。所述方法可以进一步包含检测由所述扩增的DNA片段产生的荧光信号,并且基于检测到的荧光信号计算所述扩增的DNA片段的数量。(Described herein are methods, compositions, and kits for library quantification and identification. Some embodiments relate to a method of library quantification. For example, the method can comprise providing a DNA fragment, amplifying the DNA fragment by polymerase chain reaction PCR in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated from the amplified DNA fragments, and calculating the number of the amplified DNA fragments based on the detected fluorescent signal.)

1. A method of library quantification, the method comprising:

Providing a DNA fragment;

Amplifying said DNA fragments by polymerase chain reaction PCR in the presence of at least one primer, wherein at least one primer is labeled with a fluorophore resulting in a predetermined number of fluorophores attached to each amplified DNA fragment;

Detecting a fluorescent signal generated from the amplified DNA fragments;

Calculating the number of the amplified DNA fragments based on the detected fluorescent signal; and

The amplified DNA fragments are prepared for solid phase ligation by diluting to a predetermined concentration.

2. The method of claim 1, further comprising:

Prior to detecting the signal generated by the amplified DNA fragments,

Primers not incorporated into the amplified DNA fragments were removed in preparation for fluorescent sequencing.

3. The method of claim 1, wherein the at least one primer comprises a first primer type and a second primer type, and the first primer type has a single attached fluorophore.

4. The method of claim 1, wherein a single fluorophore is attached to each DNA fragment.

5. The method of claim 2, wherein said detecting said signal produced by said amplified DNA fragments comprises detecting said fluorescent signal produced by fluorophores incorporated into said amplified DNA fragments using a fluorometer.

6. The method of claim 1, further comprising:

A standard curve is generated that indicates the relationship between the number of DNA fragments derived from the reference sample and the fluorescent signal generated by the DNA fragments.

7. The method of claim 6, wherein the calculating the number of amplified DNA fragments based on the detected signal comprises calculating the number of amplified DNA fragments based on the detected fluorescent signal and the standard curve.

8. The method of claim 1, further comprising:

A second measurement is performed to determine a characteristic of the amplified DNA fragments.

9. The method of claim 1, further comprising: the total mass of DNA in the sample is determined.

10. the method of claim 9, wherein the characteristics of the amplified DNA fragments comprise an average size of the amplified DNA fragments derived from a ratio between the number of fragments and DNA mass.

11. The method of claim 1, wherein the DNA fragments comprise at least one adaptor and the primer is complementary to the adaptor.

12. A nucleic acid library comprising DNA fragments each linked only to a predetermined number of fluorophores such that the number of DNA fragments is calculated based on fluorescent signals generated by the linked fluorophores.

13. The nucleic acid library of claim 12, wherein the DNA fragments are PCR amplicons generated using two different primers, and wherein one primer is labeled with a fluorophore such that only a predetermined number of fluorophores are attached to each PCR amplicon fragment.

14. The nucleic acid library of claim 12, wherein the DNA fragments comprise adapters and the primers are complementary to the adapters.

15. A method of sequencing a DNA sample, the method comprising:

Generating DNA fragments using the DNA sample;

Amplifying said DNA fragments by polymerase chain reaction PCR in the presence of two different primers, wherein one primer is labeled with a fluorophore to provide a predetermined number of fluorophores attached to each DNA fragment;

detecting a fluorescent signal generated from the amplified DNA fragments;

Calculating the number of the amplified DNA fragments based on the detected fluorescent signal,

Diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing; and

Sequencing at least a portion of the amplified DNA fragments using fluorescence-based sequencing techniques.

16. The method of claim 15, further comprising:

Prior to detecting the signal generated by the amplified DNA fragments,

Primers not incorporated into the amplified DNA fragments were removed in preparation for fluorescent sequencing.

17. The method of claim 15, wherein only a single fluorophore is attached to each DNA fragment.

18. The method of claim 16, wherein said detecting said signal produced by said amplified DNA fragments comprises detecting said fluorescent signal produced by fluorophores incorporated into said amplified DNA fragments using a fluorometer.

19. The method of claim 18, further comprising:

Generating a standard curve indicating the relationship between the number of DNA fragments derived from the standard library and the fluorescent signal generated by said DNA fragments.

20. The method of claim 19, wherein the calculating the number of amplified DNA fragments based on the detected signal comprises calculating the number of amplified DNA fragments based on the detected fluorescent signal and the standard curve.

21. The method of claim 15, further comprising:

determining the identity of the amplified DNA fragment.

22. The method of claim 21, wherein the characteristic of the amplified DNA fragments comprises an average size of the amplified DNA fragments.

23. The method of claim 15, wherein the DNA fragments comprise an adaptor and the primer is complementary to the adaptor.

24. A kit, comprising:

two different primers complementary to the library adaptors, wherein at least one primer is labeled with a fluorophore such that the DNA fragments amplified using the primers have a predetermined number of attached fluorophores.

25. The kit of claim 24, further comprising one or more polymerases.

26. The kit of claim 24, further comprising reagents for amplification.

27. The kit of claim 24, further comprising reagents for sequencing.

28. The kit of claim 24, further comprising written instructions for using the kit.

29. The kit of claim 24, further comprising dATP, dCTP, dGTP, dTTP, or any mixture thereof.

30. The kit of claim 24, further comprising an adaptor capable of ligating to the DNA fragment.

Background

The next generation DNA sequencers typically use DNA fragments with known sequence ends. Having known sequences at both ends enables the DNA fragment to be amplified, immobilized, and provides a starting location for sequencing (e.g., a priming site). The ends of known sequences are commonly referred to as adaptors; which adjusts the DNA fragments according to the requirements of the sequencer. Not all DNA fragments in solution have adapters at each end for sequencing. PCR amplification steps using two different primers, each specific for one of the adaptors, are commonly used to enrich for fragments with two different adaptors at the ends. This collection of DNA fragments terminated with adapters is commonly referred to as a library. These libraries can be immobilized on a solid support such that the spatial distance between the library elements (e.g., adaptors between which different DNA fragments are inserted) allows visualization (detection) and identification of individual elements from each other.

the distance between elements becomes more critical because individual elements must be amplified on the surface to increase their number and allow efficient detection of fluorophores when fragments are sequenced. This amplification is commonly referred to as bridge amplification and produces what is commonly referred to as a cluster. As the fragments are amplified, clusters of fragments having the same sequence are produced on the support.

for the sequence of the DNA to be determined in a cluster, the cluster is homogeneous and does not contain DNA from any other library element. If clusters are too close together or overlap in extreme cases, it may be difficult for image analysis software to distinguish the boundaries of the clusters and combine them into a single feature for data extraction. Since the data for this cluster is from two different DNA fragments with two different sequences, the software may not be able to determine the sequences accurately. If the clusters are further apart, each cluster can be analyzed separately and the sequence determined accurately. If the clusters are too far apart, sequencing becomes inefficient. The cost of processing the sample is fixed, but the cost per cluster increases.

Since the spacing of the clusters is determined by the concentration of individual library elements, it is necessary to accurately determine the concentration of these library elements.

Disclosure of Invention

Described herein are methods, compositions, and kits for library quantification and identification. Some embodiments relate to a method of library quantification. For example, the method can comprise providing a DNA fragment, amplifying the DNA fragment by Polymerase Chain Reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated from the amplified DNA fragments, and calculating the number of the amplified DNA fragments based on the detected fluorescent signal.

In some embodiments, prior to detecting the signal generated by the amplified DNA fragments, the method can further comprise removing primers that are not incorporated into the amplified DNA fragments or quenching the signal generated by primers that are not incorporated into the amplified DNA fragments.

in some embodiments, only one fluorophore is attached to each DNA fragment.

In some embodiments, the method may further comprise the step of fluorescence-based sequencing of the amplified DNA fragments.

In some embodiments, the signal generated by the amplified DNA fragments can be detected by detecting the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments using a fluorometer.

In some embodiments, the method may further comprise generating a standard curve indicating a relationship between the number of DNA fragments derived from the standard library and the fluorescent signal generated by the DNA fragments.

In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

In some embodiments, the method may further comprise diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing.

In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

In some embodiments, the characteristic of the amplified DNA fragments may comprise an average size of the amplified DNA fragments.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments relate to a nucleic acid library comprising DNA fragments each linked only to a predetermined number of fluorophores such that the number of DNA fragments is calculated based on fluorescent signals generated by the linked DNA fragments.

In some embodiments, the DNA fragments are PCR amplicons generated using primers, each primer labeled with a fluorophore, such that only a predetermined number of fluorophores are attached to each PCR amplicon fragment.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments relate to a method of sequencing a DNA sample. For example, the method can comprise generating a DNA fragment using a DNA sample, and amplifying the DNA fragment by Polymerase Chain Reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated by the amplified DNA fragments, calculating the number of amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing, and sequencing at least a portion of the amplified DNA fragments using a fluorescence-based sequencing technique.

In some embodiments, prior to detecting the signal generated by the amplified DNA fragments, the method can further comprise removing primers that are not incorporated into the amplified DNA fragments or quenching the signal generated by primers that are not incorporated into the amplified DNA fragments.

In some embodiments, only one fluorophore is attached to each DNA fragment.

In some embodiments, the signal generated by the amplified DNA fragments can be detected by detecting the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments using a fluorometer.

In some embodiments, the method may further comprise generating a standard curve indicating a relationship between the number of DNA fragments derived from the standard library and the fluorescent signal generated by the DNA fragments.

In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

In some embodiments, the characteristic of the amplified DNA fragments may comprise an average size of the amplified DNA fragments.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments may further comprise a kit comprising an adaptor capable of ligating to a DNA fragment and a primer complementary to the adaptor. Each primer may be labeled with a fluorophore such that the DNA fragments are amplified using the primers to link each fragment to only a predetermined number of fluorophores.

in some embodiments, the kit may comprise one or more polymerases.

In some embodiments, the kit may comprise reagents for amplification.

In some embodiments, the kit may comprise reagents for sequencing.

In some embodiments, the kit may comprise written instructions for using the kit.

In some embodiments, the kit can comprise dATP, dCTP, dGTP, dTTP, or any mixture thereof.

Drawings

The detailed description is made with reference to the accompanying drawings. In the drawings, the left-most digit or digits of a reference number identify the drawing in which the reference number first appears. The use of the same reference symbols in different drawings indicates similar or identical items.

figure 1 shows an example of library quantification.

Figure 2 shows another example of library quantification.

Figure 3 shows yet another example of library quantification.

Detailed Description

Described herein are methods, compositions, and kits for library quantification and identification. Embodiments of the present disclosure relate to the surprising discovery that attaching fluorescent dyes to DNA fragments for library quantification and identification does not interfere with subsequent sequencing. In some embodiments, primers with attached fluorescent dyes are used for library quantification and identification. When the primer with the attached fluorescent dye remains in the library, the library can be subsequently sequenced using fluorescence-based sequencing techniques.

Various methods have been reported for quantifying NGS libraries. Some methods rely on electrophoretic separation of library elements and quantification of fragments of different lengths (area under the curve assessment). Bioanalyzers (agilent technologies) are commonly used in this method. In some cases, scientists may estimate the mass of fragments of a given size and apply correction factors based on their experience to determine the extent to which to dilute the library to bring it within the proper range of the sequencer. Some use this information in combination with total nucleic acid mass determined by uv spectrophotometry, again empirically to derive a correction factor. Still others use qPCR to more accurately determine the mass of the actual library (rather than the total nucleic acid) and use this mass in conjunction with the size of the fragments measured by the bioanalyzer to more accurately determine the number of clustered units and how to appropriately dilute the sample to obtain the desired concentration for application to the DNA sequencer.

While these methods can be effective, they rely on learning/judgment, estimation, and/or cost and time consuming qPCR and bioanalyzer processing. The library contains fragments of varying lengths and length information is crucial to determining the number of fragments of a given assay mass. Thus, the accuracy of these methods may vary from individual to individual and from library to library. For example, if the size of the clustered elements is half of the estimated value, the concentration may be twice the expected value. Furthermore, since not all fragments present in solution have two adaptors or two different adaptors, these fragments can mask the true clustering elements and lead to inaccurate average size estimates.

The present disclosure provides techniques for determining the number of elements that can produce a cluster. Some embodiments of the present disclosure relate to a method for library quantification and identification that does not rely on techniques such as uv spectrophotometry, qPCR, and mean fragment size estimation.

in some embodiments, fluorescently labeled PCR primers can be used for the library enrichment step. Because the enrichment step produces amplicons with a single fluorophore or a predetermined number of fluorophores per fragment, the number of molecules can be determined. Each amplification product (e.g., amplicon) can have a predetermined number of fluorophores regardless of its length. For example, only those elements that have been amplified have fluorophore binding. After PCR enrichment, unincorporated primers were removed and the amount of fluorescent primers/amplicons was determined by fluorescence. A standard curve can be generated to determine the amount of fluorescent fragments in solution.

Unless otherwise indicated, the terms and symbols of biochemistry, nucleic acid chemistry, molecular biology, and molecular genetics follow those of standard papers and texts in the art.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" can refer to one reagent or a mixture of such reagents, and reference to "the method" includes reference to the same step and/or methods known to those skilled in the art, and so forth.

The term "adaptor" as used herein may refer to an oligonucleotide of known sequence, which is linked to a specific nucleic acid sequence or target polynucleotide strand of interest capable of producing an amplification-ready product of the specific nucleic acid or the target polynucleotide strand of interest. The specific nucleic acid sample may or may not be fragmented prior to the addition of the at least one adaptor.

Various adaptor designs are contemplated that are suitable for generating amplification-ready products for a particular sequence region/strand of interest. For example, when a double-stranded adaptor is used, the two strands of the adaptor may be self-complementary, non-complementary, or partially complementary. The adapter may contain at least a portion of the forward sequence priming site and a random sequence.

As used herein, the terms "amplifying", "amplifying" and "amplifying" used herein for a particular nucleic acid may refer to the process of producing multiple copies of a nucleic acid sample of interest, for example in the form of DNA copies. Many methods and protocols for amplifying nucleic acids are known in the art, such as PCR and qPCR.

As used herein, the term "cDNA" as used herein may refer to complementary DNA. DNA can be synthesized from messenger rna (mrna) templates in reactions catalyzed by reverse transcriptase and DNA polymerase.

As used herein, the term "complementary" as used herein may refer to complementary to all or only a portion of a sequence. The number of nucleotides in the hybridizable sequence of the specific oligonucleotide primer or probe can be such that the stringency conditions used to hybridize the oligonucleotide primer or probe can prevent excessive random non-specific hybridization. The number of nucleotides in the hybridizing portion of the oligonucleotide primer or probe can be at least as great as the defined sequence of the target polynucleotide to which the oligonucleotide primer or probe hybridizes, typically from about 20 to about 50 nucleotides. The target polynucleotide/oligonucleotide may be larger than the oligonucleotide primer, primer or probe.

As used herein, the term "denaturing" as used herein may refer to separating double-stranded nucleic acids into single strands. Denaturation can be accomplished using any method known in the art, including but not limited to physical denaturation, thermal denaturation, and/or chemical denaturation.

As used herein, the phrase "genomic DNA" as used herein may refer to chromosomal DNA, abbreviated gDNA to represent genomic deoxyribonucleic acid. gDNA contains the genetic material of an organism.

As used herein, the term "genome" as used herein may refer to DNA, RNA or cDNA sequences derived from a patient, tissue, organ, single cell, tumor, organic fluid sample taken from a patient, free-circulating nucleic acids, fungi, prokaryotes and viruses.

As used herein, the term "transcriptome" as used herein may be all RNA sequences capable of reflecting part or all of the expression genome of an organism.

As used herein, the term "kit" as used herein may refer to any system for delivering materials. In the context of reaction assays, such delivery systems may contain elements that allow for the storage, transport, or delivery of reaction components (e.g., oligonucleotides, buffer components, additives, reaction enhancers, enzymes, etc.) from one location to another in a suitable container, typically with written instructions for conducting the assay. The kit may comprise one or more housings or cassettes containing the relevant reaction reagents and support materials. The kit may comprise two or more separate containers, wherein each container comprises a portion of the total kit components. The containers may be delivered to the intended recipient together or separately.

As used herein, the phrase "Nucleic Acid (NA) modifying enzyme" as used herein may refer to a DNA-specific modifying enzyme. For the specificity of double-stranded DNA, a NA modifying enzyme may be selected. The enzyme may be a double-strand specific endonuclease, a blunt-end frequent-cleavage restriction enzyme, or another restriction enzyme.

As used herein, the phrases "nucleic acid fragment" and "specific nucleic acid" are used interchangeably, and as used herein may refer to a portion of a nucleic acid sample. Nucleic acids in an input sample can be partitioned into populations of fragmented nucleic acid molecules or polynucleotides of one or more specific size ranges.

as used herein, the phrase "specific nucleic acid sequence" or "specific sequence" as used herein may be a polynucleotide sequence of interest for which digital measurement and/or quantification is desired, including but not limited to nucleic acid fragments. The particular sequence may be known or unknown as to its actual sequence. As used herein, a "template" may be a polynucleotide comprising a particular nucleic acid sequence. The terms "specific sequence," "specific nucleic acid sequence," "specific nucleotide sequence," "region of interest," or "sequence of interest," and variants thereof, are used interchangeably.

as used herein, the phrases "qualified nucleic acid" and "identifying target nucleic acid fragments" as used herein may refer to fragments of a gDNA or RNA sequence, i.e.: i.) the acceptable template for the DNA polymerase, i.e. the template may be free of cross-linking or inhibitors of the DNA polymerase, or ii) the template has modifications, including but not limited to, ligating at the 5 'and/or 3' end at least one of a polynucleotide sequence, i.e. a barcode, an adaptor, a sequence complementary to a primer, etc., such that the fragment may be modified for quantification, amplification, detection or other methods known to those skilled in the art of gDNA and cDNA sequence analysis.

As used herein, the term "oligonucleotide" may refer to a polynucleotide chain, typically less than 200 residues in length, for example between 15 and 100 nucleotides in length, but may also include longer polynucleotide chains. The oligonucleotide may be single-stranded or double-stranded. As used in this disclosure, the term "oligonucleotide" may be used interchangeably with the terms "primer", "probe" and "adaptor".

As used herein, "PCR" is an abbreviation for the term "polymerase chain reaction" and is a commonly used nucleic acid amplification technique. In some embodiments, PCR uses two oligonucleotide primers for each designed strand, such as extension of one primer to provide a template for the other primer in the next PCR cycle. To distinguish the oligonucleotide primers in question, either of a pair of oligonucleotide primers may be designated herein as a "forward" or "reverse" primer. PCR may consist of: repeating (or cycling) (i) a denaturation step that separates strands of the double-stranded nucleic acid, followed by (ii) an annealing step that allows annealing of the primers to flanking positions of the sequence of interest; and then (iii) an extension step, which extends the primer in the 5 'to 3' direction, thereby forming a nucleic acid fragment complementary to the target sequence. Each of the above steps may be performed at different temperatures using an automated thermal cycler. The PCR cycle can be repeated as often as desired, resulting in exponential accumulation of the target DNA fragment, the end of which is generally defined by the 5' end of the primers used.

The phrase "quantitative PCR" or "qPCR" as used herein may refer to PCR designed to measure the abundance of one or more specific target sequences in a sample. Quantitative measurements can be made using one or more reference nucleic acid sequences, which can be determined alone or in combination with the target nucleic acid.

The term "portion" as used herein may refer to less than the total length of a nucleic acid sequence, a fragment of a nucleic acid sequence, a particular nucleic acid fragment, a probe, a primer, etc.

The term "primer" as used herein can refer to an oligonucleotide, typically having a free 3' hydroxyl group, that is capable of hybridizing or annealing to a template (e.g., a particular polynucleotide, target DNA, target RNA, primer extension product, or probe extension product), and is also capable of promoting polymerization of a polynucleotide that is complementary to the template. The primer may contain a non-hybridizing sequence constituting a tail portion of the primer. The primer can hybridize to the target even if the primer sequence is not completely complementary to the target.

The primers used herein may be oligonucleotides used in extension reactions, such as PCR, qPCR, extension reactions, etc., performed by a polymerase along a polynucleotide template. The oligonucleotide primer may be a synthetic polynucleotide, possibly single-stranded, containing at its 3' end a sequence capable of hybridizing to the sequence of the target polynucleotide.

The 3' region of the primer that hybridizes to a particular nucleic acid may comprise at least 80%, preferably 90%, more preferably 95%, and most preferably 100% complementarity to the sequence or primer binding site.

The term "sample" as used herein may refer to any substance that contains or is assumed to contain a nucleic acid of interest, and thus includes nucleic acids, cells, organisms, tissues, bodily fluids (e.g., spinal or lymphatic fluids), organic fluids taken from a patient, and samples including, but not limited to: blood, plasma, serum, urine, tears, feces, respiratory and genitourinary tracts, saliva, fragments of different organs, tissues, blood cells, Circulating Tumor Cells (CTCs) or disseminated tumor Cells (CTDs), bones, in vitro cell culture samples or samples suspected of containing nucleic acid molecules.

The term "PCR repeat" as used herein may refer to any sequencing read that is derived from the same original nucleic acid molecule and thus from the same primer/probe extension product sequence as another sequencing read, and thus does not represent a unique nucleic acid molecule.

Additional information regarding the definitions, processes, method structures, and other embodiments is given in U.S. patent publication No. US20160203259, assigned to nuclear age corporation (Nugen Corp.), which is incorporated by reference in its entirety.

embodiments of the present disclosure relate to methods, compositions, and kits for library quantification and identification.

some embodiments relate to a method of library quantification. In some embodiments, the method can comprise providing a DNA fragment, and amplifying the DNA fragment by Polymerase Chain Reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated from the amplified DNA fragments, and calculating the number of the amplified DNA fragments based on the detected fluorescent signal.

Some embodiments relate to methods of library quantification using two or more types of primers. Each primer type may have an associated single fluorophore, multiple fluorophores, or no fluorophores at all. For those embodiments having two types of primers, the first type will have an associated single fluorophore, and the second type of primer will have no fluorophore. The DNA fragments may be amplified by Polymerase Chain Reaction (PCR) in the presence of at least one primer, wherein the at least one primer is labeled with a fluorophore, resulting in a predetermined number of fluorophores attached to each DNA fragment. Detecting a fluorescent signal generated from the amplified DNA fragments, and calculating the number of the amplified DNA fragments based on the detected fluorescent signal. In some embodiments, the DNA fragments for fluorescent sequencing may be further prepared by diluting the amplified DNA fragments to a predetermined concentration.

In some embodiments, prior to detecting the signal generated by the amplified DNA fragments, the method can further comprise removing primers that are not incorporated into the amplified DNA fragments or quenching the signal generated by primers that are not incorporated into the amplified DNA fragments.

In some embodiments, unincorporated fluorescent PCR primers can be removed prior to performing quantitative measurements. For example, the fluorescence of the primer can be quenched. After the PCR reaction, quenching of the unincorporated dye can be achieved by annealing a short oligonucleotide, which is complementary to the fluorescent oligonucleotide and to which a compound capable of quenching the fluorophore is attached. When multiple samples are planned to be pooled together prior to sequencing, some embodiments of the disclosure may enable crude samples to be accurately quantified, mixed in appropriate proportions, and then purified as a whole rather than individually.

In some embodiments, a single oligonucleotide with a quencher can be used to measure the functional element in the crude mixture, and enrichment will be performed using an oligonucleotide with a hairpin structure and with both a fluorophore and a quencher. When the oligonucleotide is in the hairpin structure, the fluorophore and quencher are in close proximity to interfere with fluorescence detection. When the oligonucleotide structure relaxes and the oligonucleotide anneals to the PCR template, the spacing between the fluorophore and the quencher increases, allowing the fluorophore to be detected. After PCR, the hairpin structure is reformed in the unincorporated oligonucleotide when the solution is cooled. When the measurement is performed, the oligonucleotide that incorporated the amplicon is detected, but the unincorporated oligonucleotide is dark.

In some embodiments, only one fluorophore is attached to each DNA fragment.

In some embodiments, the method may further comprise the step of fluorescence-based sequencing of the amplified DNA fragments.

in some embodiments, the signal generated by the amplified DNA fragment can be detected by detecting the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragment using a fluorometer (alternatively spelled as a "fluorometer").

In some embodiments, the method may further comprise generating a standard curve indicating a relationship between DNA fragments derived from the standard library and the fluorescent signal generated by the DNA fragments.

In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

In some embodiments, the method may further comprise diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing.

In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments. For example, a fluorescent intercalating dye can be added to the sample after the measurement of the fluorescent primer. Fluorescent intercalating dyes can bind proportionally to the total mass of double stranded DNA. The absolute mass can then be determined by comparing this fluorescence reading to a standard curve. By accurate element numbers and total mass, the average size of the library fragments can be determined. The measurement can provide data correlating to the quality of the library and whether the library was formed correctly.

in some embodiments, the characteristic of the amplified DNA fragments may comprise an average size of the amplified DNA fragments.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments relate to a nucleic acid library comprising DNA fragments each linked only to a predetermined number of fluorophores such that the number of DNA fragments is calculated based on fluorescent signals generated by the linked DNA fragments.

in some embodiments, the DNA fragments are PCR amplicons generated using primers, each primer labeled with a fluorophore, such that only a predetermined number of fluorophores are attached to each PCR amplicon fragment.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments relate to a method of sequencing a DNA sample. For example, the method can comprise generating a DNA fragment using a DNA sample, and amplifying the DNA fragment by Polymerase Chain Reaction (PCR) in the presence of primers each labeled with a fluorophore. In these cases, only a predetermined number of fluorophores are attached to each DNA fragment. The method may further comprise detecting a fluorescent signal generated by the amplified DNA fragments, calculating the number of amplified DNA fragments based on the detected fluorescent signal, diluting the amplified DNA fragments to a predetermined concentration suitable for fluorescence-based sequencing, and sequencing at least a portion of the amplified DNA fragments using a fluorescence-based sequencing technique.

In some embodiments, prior to detecting the signal generated by the amplified DNA fragments, the method can further comprise removing primers that are not incorporated into the amplified DNA fragments or quenching the signal generated by primers that are not incorporated into the amplified DNA fragments.

In some embodiments, only one fluorophore is attached to each DNA fragment.

In some embodiments, the signal generated by the amplified DNA fragments can be detected by detecting the fluorescent signal generated by the fluorophore incorporated into the amplified DNA fragments using a fluorometer.

In some embodiments, the method may further comprise generating a standard curve indicating a relationship between DNA fragments derived from the standard library and the fluorescent signal generated by the DNA fragments.

In some embodiments, the number of amplified DNA fragments can be calculated based on the detected signal by calculating the number of amplified DNA fragments based on the detected fluorescent signal and a standard curve.

In some embodiments, the method may further comprise determining the identity of the amplified DNA fragments.

in some embodiments, the characteristic of the amplified DNA fragments may comprise an average size of the amplified DNA fragments.

In some embodiments, the DNA fragment may comprise an adaptor, and the primer is complementary to the adaptor.

Some embodiments may further comprise a kit comprising an adaptor capable of ligating to a DNA fragment and a primer complementary to the adaptor. Each primer may be labeled with a fluorophore such that the DNA fragments are amplified using the primers to link each fragment to only a predetermined number of fluorophores.

In some embodiments, the kit may comprise one or more polymerases.

in some embodiments, the kit may comprise reagents for amplification.

In some embodiments, the kit may comprise reagents for sequencing.

In some embodiments, the kit may comprise written instructions for using the kit.

In some embodiments, the kit can comprise dATP, dCTP, dGTP, dTTP, or any mixture thereof.

The disclosure is further described with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the disclosure should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations which become evident as a result of the teachings provided herein.

Example 1

Two lllumina TruSeq DNA libraries (BC11 and BC13) were PCR amplified using fluorescently labeled PCR primers (/56-FAM/CAA GCA GAA GAC GGC ATA CG (SEQ ID: 1)). The purified libraries were analyzed on an agilent bioanalyzer to determine the average size of the libraries, and the results are in fig. 1 (a). The library was also quantified by a NanoDrop uv-vis spectrophotometer and a KAPA library quantification kit. Molar concentrations were calculated using the amounts determined by the KAPA library quantification kit and the average size of the library determined by the bioanalyzer. 2 to 8 microliter of the library was mixed with 200. mu.L of TE buffer and the fluorescence read on a qubit 2.0 fluorometer. The linear dependence of qubit fluorescence readings on molar mass is shown in fig. 1 (B).

Two lllumina TruSeq DNA libraries (BC12 and BC14) were PCR amplified using fluorescently labeled PCR primers (/56-FAM/CAA GCA GAA GAC GGC ATA CG (SEQ ID: 1)). 5 microliter of the library was mixed with 200. mu.L of TE buffer and the fluorescence read on a qubit 2.0 fluorimeter. The library BC13 was used as a standard for calculating the molarity of the libraries BC12 and BC14, as shown in fig. 2 (a-C). An equimolar pool of four libraries was sequenced on the lllumina MiSeq sequencer. The sequencing results are in FIG. 2 (D).

Example 2

Six DNA libraries were formed by using fluorescently labeled PCR primers (/56-FAM/CAA GCA GAA GAC GGC ATA CG (SEQ ID:1)) in the final PCR amplification. By accurate element numbers and total mass, the average size of the library fragments can be determined. Accordingly, the purified library was analyzed on an agilent bioanalyzer to determine the average size of the library, the results of which are shown in fig. 3 (a). 2 microliter library with 198 u L qubit dsDNA HS reagent or 198 u L TE buffer mixed, and in the qubit 2.0 fluorometer reading fluorescence. The results are shown in table 1. The correlation of library size and the ratio of the qubit reads (DNA dye/Fam) is shown in fig. 3 (B).

TABLE 1

Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts are disclosed as example forms of the claims.

Sequence listing

<110> Netzian Technologies Inc. (NuGEN Technologies, Inc.)

<120> library quantification and identification

<130> NUGEN 00101

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis

<400> 1

caagcagaag acggcatacg 20