阅读说明：本技术 检测样品中生物靶标的空间分布的方法和系统 (Method and system for detecting spatial distribution of biological targets in a sample ) 是由 马克·S·朱 大卫·罗腾伯格 于 2014-06-25 设计创作，主要内容包括：本发明提供了运用于空间编码生物分析的方法和分析系统,包括检测样品中多个位点的一个或者多个生物靶标的丰度、表达和/或活性的空间分布的分析。尤其是,所述生物靶标是核酸,本发明的方法和分析系统不依赖于用于获取靶标空间信息的成像技术。本发明提供了在其中试剂被提供给生物样品用来标记试剂被传送到的位点的高水平多重分析的方法和分析系统；能够控制试剂传送的仪器；及提供数字化读数的解码方案。(The present invention provides methods and assay systems for use in spatially encoded biological assays, including assays that detect the spatial distribution of abundance, expression, and/or activity of one or more biological targets at multiple sites in a sample. In particular, where the biological target is a nucleic acid, the methods and analysis systems of the present invention do not rely on imaging techniques for obtaining spatial information about the target. The present invention provides methods and assay systems in which reagents are provided to a biological sample for high level multiplex analysis of the site to which the reagents are delivered; an instrument capable of controlling reagent delivery; and a decoding scheme that provides digitized readings.)

1. A method of determining a spatial distribution of abundance, expression and/or activity of a biological target in a sample, wherein the biological target is a nucleic acid, the method comprising:

(a) attaching a sample to at least one microfluidic device having a plurality of addressing channels;

(b) delivering a probe for the nucleic acid to a site in a sample, the probe comprising:

(i) a target binding moiety capable of binding to the nucleic acid; and

(ii) an address tag that identifies each of a plurality of sites to which the probe is delivered;

(c) binding each probe to the nucleic acid in the sample;

(d) analyzing the probe bound to the nucleic acid: (1) determining the abundance, expression, and/or activity of the nucleic acid by assessing the amount of the probe bound to the nucleic acid; and (2) determining the identity of the address tag of the probe; and

(e) determining a spatial distribution of the abundance and/or activity of the nucleic acids at a plurality of sites in the sample based on the analysis.

2. A method of determining a spatial distribution of abundance, expression and/or activity of a biological target in a sample, wherein the biological target is a nucleic acid, the method comprising:

(a) delivering a probe for the nucleic acid to a site in a sample, the probe comprising:

(i) a target binding moiety capable of binding to the nucleic acid; and

(ii) an address tag that identifies each of a plurality of sites to which the probe is delivered;

(b) binding each probe to the nucleic acid in the sample;

(c) analyzing the probe bound to the nucleic acid: (1) determining the abundance, expression, and/or activity of the nucleic acid by assessing the amount of the probe bound to the nucleic acid; and (2) determining the identity of the address tag of the probe; and

(d) determining a spatial distribution of the abundance and/or activity of the nucleic acids at a plurality of sites in the sample based on the analysis.

3. A method of analyzing a biological target in a sample, wherein the biological target is a nucleic acid, the method comprising:

(a) delivering probes for said nucleic acids to a plurality of sites in a sample, said probes comprising a binding moiety capable of specifically binding to said nucleic acids;

(b) delivering an address tag to a site of the plurality of sites in the sample, wherein the address tag is for binding to the probe and identifying the site in the sample; and

(c) analyzing the probe/address tag conjugate by sequencing, wherein the sequencing product comprises all or part of the address tag.

4. A method of analyzing a biological target in a sample, wherein the biological target is a nucleic acid, the method comprising:

(a) delivering probes for said nucleic acids to a plurality of sites in a sample, said probes comprising a binding moiety capable of specifically binding to said nucleic acids;

(b) delivering a first address tag to a first site in the sample, wherein the first address tag is for binding to the probe and identifying the first site in the sample;

(c) delivering a second address tag to a second site in the sample, wherein the second address tag identifies the second site in the sample and the second address tag is used to bind the conjugate of the probe and the first address tag at the intersection of the first site and the second site in the sample; and

(d) analyzing the binding of the probe, the first address tag, and the second address tag by sequencing, wherein the sequencing product comprises all or part of the first address tag and the second address tag.

5. A system for determining a spatial distribution of abundance, expression, and/or activity of one or more biological targets in a sample, wherein the one or more biological targets are one or more nucleic acids, the system comprising:

a first module for delivering a probe for each of one or more nucleic acids to a plurality of sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the respective nucleic acid;

a second module for delivering an address tag to each of the plurality of sites in the sample, wherein the address tag is for binding to a probe that binds to the nucleic acid and identifying the site to which the address tag is delivered;

a third module for analyzing probe/address tag conjugates bound to the one or more nucleic acids, the analysis comprising: (1) determining the abundance, expression, and/or activity of the one or more nucleic acids by assessing the amount of the probe/address tag conjugate bound to the one or more nucleic acids; and (2) determining the identity of the identification tag and the address tag in the probe/address tag conjugate; and

a fourth module for determining a spatial distribution of abundance, expression, and/or activity of the one or more nucleic acids at a plurality of sites in the sample based on the analysis.

6. The system of claim 5, wherein the second module comprises one or more microfluidic devices for communicating the address tag.

7. The system of claim 6, wherein the one or more microfluidic devices comprise a first set of composite addressing channels and a second set of composite addressing channels, wherein each of the first set of composite addressing channels delivers a different first address label to the sample and each of the second set of composite addressing channels delivers a different second address label to the sample.

8. A method of determining a spatial distribution of abundance, expression and/or activity of a biological target in a sample, comprising:

(a) delivering a probe for the biological target to a site in a sample, the probe comprising:

(i) a target binding moiety capable of binding the biological target; and

(ii) an address tag that identifies each of a plurality of sites to which the probe is delivered;

(b) binding each probe to the biological target in the sample;

(c) analyzing the probes bound to the biological target: (1) determining abundance, expression, and/or activity of the biological target by assessing the amount of the probe bound to the biological target; and (2) determining the identity of the address tag of the probe; and

(d) determining a spatial distribution of the abundance and/or activity of the biological target over a plurality of sites in the sample based on the analysis.

9. A method of determining a spatial distribution of abundance, expression and/or activity of a biological target in a sample, comprising:

(a) attaching a sample to at least one microfluidic device having a plurality of addressing channels;

(b) delivering a probe for the biological target to a site in a sample, the probe comprising:

(i) a target binding moiety capable of binding the biological target; and

(ii) an address tag that identifies each of a plurality of sites to which the probe is delivered;

(c) binding each probe to the biological target in the sample;

(d) analyzing the probes bound to the biological target: (1) determining abundance, expression, and/or activity of the biological target by assessing the amount of the probe bound to the biological target; and (2) determining the identity of the address tag of the probe; and

(e) determining a spatial distribution of the abundance and/or activity of the biological target over a plurality of sites in the sample based on the analysis.

10. A method of analyzing a biological target in a sample, comprising:

(a) delivering a probe for a biological target to a plurality of sites in a sample, the probe comprising a binding moiety capable of specifically binding to the biological target;

(b) delivering an address tag to a site of the plurality of sites in the sample, wherein the address tag is for binding to the probe and identifying the site in the sample;

(c) analyzing the probe/address tag conjugate by sequencing, wherein the sequencing product comprises all or part of the address tag.

11. A method of analyzing a biological target in a sample, comprising:

(a) delivering a probe for a biological target to a plurality of sites in a sample, the probe comprising a binding moiety capable of specifically binding to the biological target;

(b) delivering a first address tag to a first site in the sample, wherein the first address tag is for binding to the probe and identifying the first site in the sample;

(c) delivering a second address tag to a second site in the sample, wherein the second address tag identifies the second site in the sample and the second address tag is used to bind the conjugate of the probe and the first address tag at the intersection of the first site and the second site in the sample;

(d) analyzing the binding of the probe, the first address tag, and the second address tag by sequencing, wherein the sequencing product comprises all or part of the first address tag and the second address tag.

12. A system for determining a spatial distribution of abundance, expression, and/or activity of one or more biological targets at a plurality of sites in a sample, comprising:

a first module for delivering a probe for each of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises a target-binding portion capable of binding a respective biological target of the probes;

a second module for delivering an address tag to each of the plurality of sites in the sample, wherein the address tag is for binding to a probe that binds to the biological target and identifying the site to which the address tag is delivered;

a third module for analyzing probe/address tag conjugates bound to the one or more biological targets, the analysis comprising: (1) determining abundance, expression, and/or activity of the one or more biological targets by assessing the amount of the probe/address tag conjugate bound to the biological target; and (2) determining the identity of the identification tag and the address tag in the probe/address tag conjugate; and

a fourth module for determining a spatial distribution of abundance, expression, and/or activity of the one or more biological targets at a plurality of sites in the sample based on the analysis.

13. The system of claim 12, wherein the second module comprises one or more microfluidic devices for communicating the address tag.

14. The system of claim 13, wherein the one or more microfluidic devices comprise a first set of composite addressing channels and a second set of composite addressing channels, wherein each of the first set of composite addressing channels delivers a different first address label to the sample and each of the second set of composite addressing channels delivers a different second address label to the sample.

15. The method of any of claims 1-4 or 8-11, further comprising: hybridizing the probe to the biological target between steps (a) and (b).

16. The method of any of claims 1-4, 8-11, or 15, further comprising: extending the probe using the biological target as a template, thereby generating an oligonucleotide sequence comprising at least a portion of the probe and at least a portion of the biological target.

17. The method of any one of claims 1-4, 8-11, 15, or 16, wherein the probe further comprises a first linking region and the address tag comprises a second linking region, the probe being bound to the address tag by a link between the first linking region and the second linking region.

18. The method of any one of claims 4, 11 or 15-17, wherein the first address tag and the second address tag located at the intersection of the first site and the second site in the sample are the same.

19. The method of any one of claims 4, 11 or 15-17, wherein the first address tag and the second address tag located at the intersection of the first site and the second site in the sample are different.

20. The method of any one of claims 1-4, 8-11, or 15-19, wherein the probe comprises a plurality of binding moieties capable of specifically binding to the same sequence or to different sequences in the biological target, or capable of specifically binding to different biological targets.

21. The method of any one of claims 1-4, 8-11, or 15-20, wherein the sample is a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

22. The method of any one of claims 1-4, 8-11, or 15-21, wherein the address tag comprises an oligonucleotide.

23. The method of any one of claims 1-4, 8-11, or 15-22, wherein the analysis is performed by high throughput digital nucleic acid sequencing.

24. The method of any one of claims 1-4, 8-11, or 15-23, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 20.

25. The method of any one of claims 1-4, 8-11, or 15-24, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 50.

26. The method of any one of claims 1-4, 8-11, or 15-25, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 75.

27. The method of any one of claims 1-4, 8-11, or 15-26, wherein the product of the number of biological targets to be assayed in the sample and the number of sites to be assayed is greater than 100.

28. The method of any one of claims 1-4, 8-11, or 15-27, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 1,000.

29. The method of any one of claims 1-4, 8-11, or 15-28, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 10,000.

30. The method of any one of claims 1-4, 8-11, or 15-29, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 100,000.

31. The method of any one of claims 1-4, 8-11, or 15-30, wherein the product of the number of biological targets to be assayed and the number of sites to be assayed in the sample is greater than 1,000,000.

32. The method of any one of claims 1-4 or 8-11, wherein at least one hundred thousand probes or probe/address label conjugates are assayed in parallel.

33. The method of claim 32, wherein at least fifty thousand probes or probe/address label conjugates are analyzed in parallel.

34. The method of claim 33, wherein at least one million probes or probe/address label conjugates are analyzed in parallel.

35. The method of any one of claims 1-4 or 8-11, wherein a known percentage of probes for the nucleic acid are attenuation probes.

36. The method of claim 35, wherein the attenuation probe limits the yield of amplification products.

37. The method of claim 35, wherein the attenuating probe lacks 5' phosphate.

38. The method of any one of claims 1-4 or 8-11, wherein the address tag is bound to the probe by ligation or by extension or any combination thereof.

Technical Field

The present application relates generally to the analysis of biomolecules and, in particular, to methods, compositions and analytical systems for detecting the spatial distribution of abundance, expression and/or activity of one or more biological targets in a sample.

Background

In the discussion that follows, specific articles and methods are described for background introduction purposes. Nothing contained herein is to be construed as an "admission" of prior art. The applicant reserves the right in particular to make the following proof in due course: the articles and methods cited herein do not constitute prior art, in accordance with applicable legal provisions.

Integrated gene expression analysis and protein analysis have become useful tools for understanding biological mechanisms. Using these tools, genes and proteins involved in developmental processes and in various types of diseases (e.g., cancer and autoimmune diseases) can be identified. Traditional methods, such as in situ hybridization of different transcripts and other multiplex assays, have revealed spatial distribution of gene expression and help elucidate the molecular basis of development and disease. Other techniques that can be used to quantify multiple RNA sequences in each sample include microarrays (see, e.g., Shi et al, Nature Biotechnology,24(9):1151-61 (2006); and Slonim and Yanai, Ploss Computational Biology,5(10): e1000543 (2009)); gene expression continuity analysis (SAGE) (see Velculescu et al, Science,270(5235):484-87 (1995)); high throughput operation of qPCR (see Spurgeon et al, Plos ONE,3(2): e1662 (2008)); in situ PCR (see Nuovo, Genome Res.,4:151-67 (1995)); and transcriptome sequencing technology (RNA-seq) (see Mortazavi et al, Nature Methods,5(7):621-8 (2008)). While these methods are useful, they do not simultaneously measure the expression of multiple genes or the presence and/or activity of multiple proteins at multiple spatial locations in a sample.

Certain 2D forms of PCR analysis preserve spatial information (see Armani et al, L ab on a chip,9(24):3526-34(2009)) but these methods have low spatial resolution because they rely on physical transfer of tissue to reaction wells, which also prevents random access to (pending) tissue samples and high levels of multiplication.

Currently, there is a need to simultaneously analyze the spatial expression pattern of a large number of genes, proteins, or other biologically active molecules at high resolution. It is also necessary to analyze reproducible, high-resolution spatial maps of biomolecules in tissue. The disclosure of the present application meets these needs.

Disclosure of Invention

In one aspect, the present application discloses a method of detecting a spatial distribution of abundance, expression and/or activity of one or more biological targets at a plurality of sites in a biological sample, comprising:

delivering a probe for each of the one or more biological targets to a plurality of sites in the sample, wherein each probe comprises: (1) a target binding moiety capable of binding to a corresponding biological target of the probe; (2) an address tag that identifies each of the plurality of sites to which the probe is delivered; and (3) an identification tag that identifies the corresponding biological target or target-binding portion of the probe;

allowing each probe to bind to its corresponding biological target in the sample;

analyzing the probes bound to the one or more biological targets, the analyzing comprising: (1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of the probe bound to the biological target; and (2) determining the identity of the identification tag and the address tag of the probe; and

determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In some embodiments, the method does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample. In one embodiment, the analysis of probes bound to one or more of the biological targets can be accomplished using sequencing, wherein the number of sequencing products indicates the abundance, expression, and/or activity of each of the one or more of the biological targets, and the sequencing products can include all or part of the sequence of the address tag and all or part of the identification tag sequence.

In another aspect, the present application discloses a method of determining a spatial distribution of abundance, expression, and/or activity of one or more biological targets at a plurality of sites in a sample, comprising:

delivering a probe for each of the one or more biological targets to a plurality of sites in the sample, wherein each probe comprises: (1) a target binding moiety capable of binding to a corresponding biological target of the probe; and (2) an identification tag for identifying the corresponding biological target or target-binding portion of the probe;

allowing each probe to bind to its corresponding biological target in the sample;

transmitting an address tag to each of the plurality of sites in the sample, wherein the address tag is for pairing with the probe that binds to the biological target and identifying the site to which the address tag is delivered;

analyzing the probe/address tag conjugates bound to the one or more biological targets, the analyzing comprising: (1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of the probe/address tag conjugate bound to the biological target; and (2) determining the identity of the identification tag and the address tag in the probe/address tag conjugate; and

determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In some embodiments, the method does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample. In one embodiment, sequencing may be employed to analyze the probe/address tags bound to one or more of the biological targets, wherein the number of sequencing products is indicative of the abundance, expression, and/or activity of each of the one or more biological targets, and the sequencing products may include all or part of the address tag sequence and all or part of the identification tag sequence. .

In any of the preceding embodiments or any combination thereof, the one or more biological targets may be non-nucleotide molecules. In any of the preceding embodiments, the one or more biological targets may include a protein, a lipid, a carbohydrate, or any combination thereof. In any of the preceding embodiments, there are at least two address tags identifying each of the plurality of sites in the sample.

In any of the preceding embodiments, the spatial distribution of abundance, expression, and/or activity of a plurality of biological targets may be detected in parallel, and the address tag or combination of address tags on each of the plurality of biological targets may be the same at a given site in the plurality of sites in the sample. In any of the foregoing embodiments, the analyzing steps may be performed in parallel in the same reaction process.

In any of the preceding embodiments or any combination thereof, the one or more biological targets may include at least one known marker of the sample, e.g., a tissue-specific marker, a cell type marker, a cell line marker, a cell morphology marker, a cell cycle marker, a cell necrosis marker, a developmental stage marker, a stem or progenitor cell marker, a differentiation state marker, a phenotypic marker, a physiological marker, or a pathophysiological marker, a transformation state marker, a cancer marker, or a combination thereof.

In other embodiments, the present application provides a method of determining a spatial distribution of abundance, expression, and/or activity of a target protein at a plurality of sites in a sample, comprising:

delivering probes for the target protein to a plurality of sites in the sample; wherein the probe comprises: (1) a target binding moiety capable of binding to the target protein; (2) a first address tag that identifies each of the plurality of sites to which the probe is delivered, and (3) an identification tag that identifies the target protein or the target binding moiety;

allowing said probe to bind to said target protein in said sample;

analyzing probes bound to the target protein, the analyzing comprising: (1) determining the abundance, expression, and/or activity of the target protein by assessing the amount of the probe bound to the target protein; and (2) determining the identity of the identification tag and the first address tag for the target protein; and

determining a spatial distribution of abundance, expression, and/or activity of the target protein at the plurality of sites in the sample based on the analysis.

In any of the preceding embodiments, the method may further comprise:

delivering a probe for a target polynucleotide to each of the plurality of sites in the sample, wherein the probe for a target polynucleotide comprises: (1) a sequence for hybridizing to and identifying the target polynucleotide; (2) a second address tag that identifies each site of the plurality of sites to which a probe for a target polynucleotide is delivered;

allowing a probe for a target polynucleotide to bind to the target polynucleotide in the sample;

analyzing probes bound to a target polynucleotide, the analyzing comprising: (1) determining the abundance, expression, and/or activity of the target polynucleotide by assessing the amount of probe bound to the target polynucleotide; and (2) determining the identity of the sequence used to hybridize to and identify the target polynucleotide and the second address tag of the probe used for the target polynucleotide; and is

Determining a spatial distribution of abundance, expression, and/or activity of the target polynucleotide at each of the plurality of sites in the sample based on analysis of probes bound to the target polynucleotide at each of the plurality of sites in the sample.

In another aspect, the present application discloses a method of determining a spatial distribution of abundance, expression, and/or activity of a target protein at a plurality of sites in a sample, comprising:

delivering a probe for a target protein to a plurality of sites of a sample, wherein the probe comprises: (1) a target binding moiety capable of binding to the target protein; and (2) an identification tag that recognizes the target protein or the protein binding portion;

allowing said probe to bind to said target protein in said sample;

delivering a first address tag to each of the plurality of sites of the sample, wherein the first address tag is for binding to the probe bound to the target protein and identifying the site to which the probe is delivered,

analyzing the probe/first address tag conjugate bound to the target protein, the analyzing comprising: (1) determining the abundance, expression, and/or activity of the target protein by assessing the amount of the probe/first address tag binding to the target protein; and (2) determining the identity of a first address tag in the identification tag and the probe/first address tag conjugate; and

determining a spatial distribution of the abundance, expression, and/or activity of the target protein over the plurality of sites in the sample based on the analysis.

In any of the preceding embodiments, the method further comprises:

delivering a probe for a target polynucleotide to each of the plurality of sites in the sample, wherein the probe for the target polynucleotide comprises a sequence for hybridizing and recognizing the target polynucleotide.

Allowing a probe for the target polynucleotide to bind to the target polynucleotide in the sample;

transmitting a second address tag to each of the plurality of sites in the sample, wherein the second address tag is used to bind to the probe that binds to the target polynucleotide and identify the site to which it is delivered;

analyzing probe/second address tag conjugates bound to a target polynucleotide, the analyzing comprising: (1) determining the abundance, expression, and/or activity of the target polynucleotide by assessing the amount of probe/second address tag conjugate bound to the target polynucleotide; and (2) determining the identity of the sequence used to hybridize and recognize the target polynucleotide and the identity of the second address tag binding in the probe/second address tag conjugate used for the target polynucleotide; and is

Determining a spatial distribution of abundance, expression, and/or activity of the target polynucleotide at each of the plurality of sites in the sample based on an analysis of probe/second address tag conjugates bound to the target polynucleotide at each of the plurality of sites in the sample.

In one embodiment, the target polynucleotide or its complement can encode the entire target protein or a portion thereof. In some embodiments, the step of analyzing the probe or probe/first address tag conjugate bound to the target protein and the step of analyzing the probe or probe/second address tag conjugate bound to the target protein may be performed in parallel in one reaction process. In other aspects, the first address tag and the second address tag can be the same for a given site in the plurality of sites in the sample. In further aspects, the first address tag and the second address tag can be different for a given site in the plurality of sites in the sample. In any preceding embodiment, the method further comprises correlating the abundance, expression, and/or activity of the target protein and the abundance, expression, and/or activity of the target polynucleotide at each of the plurality of sites in the sample.

In any of the preceding embodiments, or any combination thereof, the biological target or target protein may comprise an enzymatic activity. In some aspects, the target binding portion of the probe of any of the preceding embodiments can comprise an antibody or antigen-binding fragment of an antibody, an aptamer, a small molecule, an enzyme substrate, a putative enzyme substrate, an affinity capture agent, or a combination thereof.

In any of the preceding embodiments or any combination thereof, the target-binding moiety binds to a polynucleotide comprising the identification tag. In any of the foregoing embodiments, the target-binding moiety can bind to a polynucleotide that specifically hybridizes to a polynucleotide comprising the identification tag. In some aspects, the probe can include multiple target-binding moieties capable of binding to the same domain or different domains of the target, or capable of binding to different targets.

In any of the preceding embodiments, or any combination thereof, the sample may be a biological sample selected from the group consisting of a fresh isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

In any of the preceding embodiments, or any combination thereof, there may be two of the address tags identifying each of the plurality of sites in the sample. In some aspects, two probes for each target may be delivered to the sample.

In any of the preceding embodiments, or any combination thereof, the address tag may comprise an oligonucleotide. In another aspect, the identification tag in any of the preceding embodiments can comprise an oligonucleotide.

In any of the preceding embodiments, or any combination thereof, the analyzing step can employ a nucleic acid sequencing technique. In one aspect, the analyzing step can employ high throughput digital nucleic acid sequencing techniques.

In any of the preceding embodiments, or any combination thereof, the results of the number of targets analyzed and the number of the plurality of sites analyzed in the sample can be greater than 20, greater than 50, greater than 75, greater than 100, greater than 1,000, greater than 10,000, greater than 100,000, or greater than 1,000,000.

In any of the preceding embodiments, or any combination thereof, at least 100,000, at least 500,000, or at least 1,000,000 probes or probe/address tag conjugates that bind to the target can be analyzed in parallel.

In any of the preceding embodiments, or any combination thereof, at least two of the transmitting step, the analyzing step, and the determining step are performed using software-programmed hardware. In any of the preceding embodiments, or any combination thereof, the step of transporting is performed using one or more microfluidic devices.

In any of the preceding embodiments, or any combination thereof, a known percentage of the probes for the biological target, the target protein, or the target polynucleotide may be attenuating probes. In one aspect, the attenuation probe can limit the yield of amplification product. For example, the attenuating probe may compete with the active probe for binding to the target. When the active probe results in the production of amplification products of the target, the attenuating probe does not reduce or has reduced the ability to produce amplification products. In one embodiment employing nucleic acid probes, the attenuator probes may lack a 5' phosphate.

In any of the preceding embodiments, or any combination thereof, the address tag can be attached to the probe by ligation, extension, post-extension ligation, or any combination thereof.

In any of the preceding embodiments, or any combination thereof, the method can further comprise constructing a three-dimensional map of the abundance, expression, and/or activity of each target from the spatial distribution of the abundance, expression, and/or activity of each target of the plurality of samples. In one aspect, the plurality of samples may be serial tissue sections of a three-dimensional tissue sample.

In another aspect, the present application provides a system for determining a spatial distribution of abundance, expression, and/or activity of one or more biological targets at a plurality of sites in a sample, comprising:

a first module for delivering probes of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises: (1) a target binding moiety capable of binding to a corresponding biological target of the probe; and (2) an identification tag for identifying the corresponding biological target or target-binding portion of the probe;

a second module for delivering an address tag to each of the plurality of sites in the sample, wherein the address tag is for binding to a probe that binds to a biological tag and identifying the site to which the address tag is delivered;

a third module for analyzing probe/address tag conjugates bound to one or more biological targets, the analysis comprising: (1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of the probe/address tag conjugate bound to the biological target; and (2) determining the identity of the identification tag and the address tag in the probe/address tag conjugate; and

a fourth module for determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In one aspect, the system does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample.

In one embodiment, the second module may include one or more microfluidic devices for communicating the address tag. In one aspect, the one or more microfluidic devices can include a first set of complex addressing channels, each channel conveying a different first address label to the sample. In one embodiment, the one or more microfluidic devices may further comprise a second set of composite addressing channels, none of which conveys a different second address tag to the sample. In one aspect, a plurality of sites in the sample are selected by the first and second sets of composite addressing channels, respectively, in cooperation delivering a first address tag and a second address tag to each site in the plurality of sites, each site being identified by a different combination of the first and second address tags.

In another embodiment, a method is disclosed, comprising: delivering a probe for each of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to its corresponding biological target; allowing each of said probes to bind to a corresponding biological target in said sample; delivering at least one adaptor to said plurality of sites of said sample, wherein said at least one adaptor specifically binds to said probe and comprises an address tag capable of identifying each of said plurality of sites to which said at least one adaptor has been delivered, wherein said probe and/or said adaptor comprises an identification tag which identifies the corresponding biological target or target binding portion of said probe and/or said adaptor; analyzing the at least one adaptor and the probe bound to the one or more of the biological targets, the analyzing comprising: (1) determining abundance, expression, and/or activity of the biological target by assessing the amount of the at least one linker bound to the biological target; and (2) determining the identity of the identification tag and the address tag of the at least one connector; determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In one aspect, the system does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample.

In yet another embodiment, a method is disclosed, comprising: delivering a probe for each of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to its corresponding biological target; allowing each of said probes to bind to a corresponding biological target in said sample; delivering at least one adaptor to said plurality of sites of said sample, wherein said at least one adaptor specifically binds to said probe and comprises an address tag capable of identifying each of said plurality of sites to which said at least one adaptor has been delivered, wherein said probe and/or said adaptor comprises an identification tag which identifies the corresponding biological target or target binding portion of said probe and/or said adaptor; analyzing the at least one linker and the probes bound to the one or more of the biological targets using sequencing techniques, wherein the amount of sequencing product is indicative of abundance, expression, and/or activity of the biological target; the sequencing product comprises part or all of the address tag sequence and part or all of the identification tag sequence; determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In one aspect, the system does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample.

In another embodiment, a method is provided, comprising: delivering a probe for each of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to its corresponding biological target; allowing each of said probes to bind to a corresponding biological target in said sample; delivering at least one adaptor to said plurality of sites of said sample, wherein said at least one adaptor specifically binds to said probe and comprises an address tag capable of identifying each of said plurality of sites to which said at least one adaptor has been delivered, wherein said probe and/or said adaptor comprises an identification tag which identifies the corresponding biological target or target binding portion of said probe and/or said adaptor; delivering an address tag to each of the plurality of sites in the sample, wherein the address tag is for binding to the at least one linker that binds to the biological target and identifying the site to which it is delivered; analyzing the linker/address tag conjugate, the analyzing comprising: (1) determining abundance, expression, and/or activity of each of the one or more biological targets by assessing the amount of the adaptor/address tag conjugate bound to the biological target; and (2) determining the identity of the identification tag and the address tag in the linker/address tag conjugate; and is

Determining a spatial distribution of abundance, expression, and/or activity of the target polynucleotide at the plurality of sites in the sample based on the analysis. In one aspect, the method does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample. .

In yet another embodiment, a method is provided, comprising: delivering a probe for each of one or more biological targets to a plurality of sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to its corresponding biological target; allowing each of said probes to bind to a corresponding biological target in said sample; delivering at least one adaptor to said plurality of sites of said sample, wherein said at least one adaptor specifically binds to said probe and comprises an address tag capable of identifying each of said plurality of sites to which said at least one adaptor has been delivered, wherein said probe and/or said adaptor comprises an identification tag which identifies the corresponding biological target or target binding portion of said probe and/or said adaptor; delivering an address tag to each of the plurality of sites in the sample, wherein the address tag is for binding to the at least one linker that binds to the biological target and identifying the site to which it is delivered; analyzing the linker/address tag conjugate using a sequencing technique, wherein the amount of sequencing product is indicative of the abundance, expression, and/or activity of the biological target; the sequencing product comprises part or all of the address tag sequence and part or all of the identification tag sequence; determining a spatial distribution of abundance, expression, and/or activity of one or more of the biological targets at a plurality of sites in the sample based on the analysis. In one aspect, the system does not rely on image techniques to determine spatial information of one or more of the biological targets in the sample.

In any preceding embodiment, at least two adaptors are delivered to each of the plurality of sites in the sample, wherein the at least two adaptors each specifically bind to one probe that specifically binds to a biological target. In one aspect, the at least two linkers are joined, e.g., using a portion of the probe sequence as a splint for ligation.

In any of the preceding embodiments, the probe for each of the one or more biological targets may comprise an affinity moiety for the biological target and an oligonucleotide, and the adaptor may comprise an oligonucleotide.

FIG. 1 is a flow chart illustrating exemplary steps of a method for detecting the spatial distribution of abundance, expression and/or activity of one or more biological targets at multiple sites in a sample according to one embodiment of the present invention.

FIG. 2 is a flow chart illustrating exemplary steps of a method for detecting the spatial distribution of abundance, expression and/or activity of one or more biological targets at multiple sites in a sample according to one embodiment of the present invention.

FIG. 3 is a combined addressing scheme according to one embodiment of the present disclosure.

FIG. 4 is a combined addressing scheme applied to a sample according to a disclosed embodiment of the invention

FIG. 5 is a combined addressing scheme applied to a sample, according to one embodiment of the present disclosure.

FIG. 6 shows a multifunctional protein detection assay using a combined addressing scheme applied to a sample, according to various embodiments of the present invention

FIG. 7 shows a representative antibody-DNA binding structure according to certain embodiments of the present invention.

FIG. 8 is a sequential address tag scheme according to a disclosed embodiment of the invention.

Fig. 9 is a microfluidic addressing device according to one embodiment of the present disclosure.

FIG. 10 provides immunofluorescence images (FIG. 10A) and representative expression profiles (FIGS. 10B-C) generated by some embodiments of the disclosure.

FIG. 11 shows a method for reducing random errors during a sequencing step (FIG. 11A) and a typical configuration using probes with integrated X and Y address tags and variable tag region Z (Figure 11B) according to some embodiments of the present invention.

Detailed Description

The following provides a detailed description of one or more embodiments of the claimed subject matter, which when taken in conjunction with the drawings, illustrate the principles of the claimed subject matter. The claimed subject matter described herein is related to these embodiments, but is not limited to any embodiment. It should be understood that the claimed subject matter may be embodied in various forms including numerous alternatives, modifications, and equivalents. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed system, structure or manner. Numerous specific details are set forth in the following description in order to provide a thorough understanding of the present invention. The detailed description is provided for the purpose of: the examples and claimed subject matter may be practiced according to the claims without some or all of these specific details. It is to be understood that other embodiments of the invention may be utilized and structural changes may be made without departing from the scope of the claimed subject matter. For the purpose of clarity, technical material that is known in the technical fields related to the claimed subject matter has not been described in detail, and thus has not been intended to obscure the claimed subject matter.

Unless defined otherwise, all technical and scientific terms or words used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and/or reference purposes, and the content of such definitions herein should not be construed to represent a substantial difference from the commonly understood meanings in the art. Many of the techniques and methods described or referenced herein are well understood and commonly employed by those of ordinary skill in the art using conventional methodology.

All publications, including patent documents, scientific and technical literature and databases, and bibliographic and additional documents, referred to herein are incorporated by reference in their entirety for the same purpose as if each individual publication was individually incorporated by reference. If a definition set forth herein conflicts or disagrees with a definition set forth in a patent, application, published application, and other disclosure incorporated by reference, the definition set forth herein controls.

Unless otherwise stated, the examples of the present application will be applied to the general techniques described herein, organic chemistry, polymer Technology, Molecular Biology (including Recombinant Technology), cell Biology, Biochemistry and sequencing techniques, all within the general skills of those of ordinary skill in the art, these general techniques include polymer array Synthesis, polynucleotide hybridization and ligation techniques and hybridization detection techniques using tags, however, other equivalent general technical Methods are certainly available, such general Methods and descriptions can be found in standard laboratory manuals, such as Green, et al, eds, Genome Analysis, A L organic series (experiments, I-IV) (1999; Weiner, scientific, Stephens, eds, genetics: A L, Handbook (safety), PCR, research, USA, 1985, Press, PCR, Press, sample, DNA, PCR, research, No. 12, PCR, research, No. 12, No. 5, sample No. 7, plant No. 7, sample No. A, sample No. 7, sample No. A, sample No. 7, sample No. A, sample No. 7, sample No. A, sample No. 7, sample No. A, sample No. 7, sample No..

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". Thus, reference herein to "a biological target" is to one or more biological targets, and reference herein to "a method" is to include, inter alia, the recited equivalent steps and methods and/or equivalent steps and methods known to those skilled in the art of the present disclosure.

Various aspects of the claimed subject matter are presented in a range format throughout this disclosure. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as a limitation to the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges as well as individual values of that range. For example, a range of values is provided, and it is understood that each intervening value, to the extent that there is no such stated, between the upper and lower limit of that range and any other stated or intervening value in that range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. The stated ranges include one or both of the limits, and ranges outside of either or both of these limits are also included within the claimed subject matter. This applies regardless of the range.

As used herein, "individual" means any living organism, including humans and other mammals. As used herein, "subject" means an organism to which the provided compositions, methods, kits, devices, and systems can be injected or used. In one embodiment, the subject is a mammal or a cell, tissue, organ, or part of a mammal. Mammals include, but are not limited to, humans, and non-human animals, including domestic animals, sport animals, rodents, and pets.

As used herein, "biological sample" may refer to a sample obtained from a organism or virus or other macromolecule or biomolecule, including any type of cell or tissue of a subject from which nucleic acids or proteins or other macromolecules may be obtained. The biological sample may be obtained directly from a biological source or may be a biological sample obtained after processing. For example, the amplified isolated nucleic acids constitute a biological sample. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples from the above.

As used herein, "ingredient" refers to any mixture of two or more products or compounds. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

The terms "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to polymeric forms of nucleotides of any length, and may include those composed of ribonucleotides, deoxyribonucleotides, analogs or mixtures thereof, the terms refer only to the primary structure of the molecule, such that the terms include triple-stranded, double-stranded and single-stranded deoxyribonucleic acids (RNAs), as well as triple-stranded, double-stranded and single-stranded ribonucleic acids (RNAs), which also include modified forms, such as by alkylation, and/or capping modifications, and unmodified forms of polynucleic acids, in particular, the terms "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2 deoxyribo), polyribonucleotides (containing ribo), such as tRNA, rRNA, hRNA and mRNA, whether or not bound or not crossed, other types of polynucleotides having N-or C-glycosides of purines or pyrimidines, and other polymers having a normal nucleotide backbone, e.g., polyamides (e.g., polypeptides ("PNAs") and polyminonucleotides (e.g., oligonucleotides), such as, PNAs), which may be modified from an Anarayne-Virae.g., oligonucleotides "and" which have a "phosphoamidinate-or phosphoamidinate-linker moiety, such as a" or a "such as a" linker which may be linked to a linker, such as a linker, a linker which includes a linker, such as a linker which may be a linker, a linker which has a linker, a linker which may be a linker, such as a linker, a linker which may be a linker, a linker which may be used interchangeably, a linker which may be a linker, a linker which includes a linker, a linker which includes a linker which has a linker, a linker which includes a linker which has a linker, a linker which has a linker which includes a linker, a linker which has a linker, a linker which has.

It will be appreciated that the terms "nucleoside" and "nucleotide" as used herein include those groups which contain not only purine and pyrimidine bases but also heterocyclic bases which have been modified. Such modifications include methylpurine or pyrimidine, acylated purines or pyrimidines or other heterocyclic compounds. Modified nucleoside or nucleotide sugars also include modifications of sugar ligands, for example, where one or more hydroxyl groups are replaced with halogens, aliphatic groups, or groups functionalized as ethers, amines, or the like. The term "nucleotide unit" is intended to encompass nucleosides and nucleotides.

"nucleic acid probe" refers to a structure comprising a polynucleotide, as defined above, comprising a nucleic acid sequence that can bind to a corresponding target. The polynucleotide region of the probe may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.

The terms "polypeptide", "oligopeptide", "peptide", and "protein" as used herein are used interchangeably and refer to polymers of amino acids of any length, for example, at least 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000 or more amino acids in length. The polymer may be linear or branched, include modified amino acids, and may be blocked by non-amino acids. These terms also encompass an amino acid polymer that is naturally modified or intervened; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation or any other manipulation or modification, such as conjugation of a tag component. For example, included within the definition of a polypeptide as an analog comprising one or more amino acids (e.g., including unnatural amino acids, etc.), as well as other modifications known in the art.

The terms "binding agent" and "target binding moiety" as used herein refer to any agent or moiety thereof that is capable of specifically binding to a biomolecule of interest.

The biological target or biomolecule to be detected may be any biomolecule including, but not limited to, proteins, nucleic acids, lipids, carbohydrates, ions or multicomponent complexes comprising any of the above molecules. Examples of subcellular targets include organelles, e.g., mitochondria, golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, vacuoles, lysosomes, and the like. Typical nucleic acid targets include chromosomal DNA (e.g., A-DNA, B-DNA, Z-DNA), mitochondrial DNA (mtDNA), mRNA, tRNA, rRNA, hRNA, miRNA, and piRNA in various conformations.

As used herein, "biological activity" includes the in vivo activity of a compound or the physiological response resulting from administration of a compound, composition or other mixture in vivo. Thus, biological activity encompasses the therapeutic effects and pharmaceutical activities of such compounds, compositions and mixtures. Biological activity can be observed in an in vitro system for detecting or using such activity.

The term "binding" may refer to an interaction between two molecules resulting in a stable binding of one molecule close to the other. Molecular binding can be classified into the following types: noncovalent, reversible covalent, irreversible covalent. Molecules involved in molecular binding include proteins, nucleic acids, carbohydrates, fats and small organic molecules such as pharmaceutical compounds. Proteins that form stable complexes with other molecules are often referred to as receptors, while their binding molecules are referred to as ligands. Nucleic acids may also form stable complexes with themselves or with others, e.g., DNA-protein complexes, DNA-DNA complexes, DNA-RNA complexes.

The term "specifically binds" as used herein refers to the specificity of a binder, e.g., an antibody, which causes it to preferentially bind to a target, e.g., a polypeptide antigen. When referring to binding molecules (e.g., proteins, nucleic acids, antibodies or other cognate capture agents, etc.), a "specific binding" can include a binding reaction of two or more binding partners having high affinity and/or complementarity to ensure selective hybridization under specified assay conditions. Typically, specific binding will have at least three times the standard deviation of the background signal. Thus, a binding molecule binds to its specific target molecule under specified conditions, without binding to a large number of other molecules present in the sample. Recognition by a binder or antibody of a particular target in the presence of other potentially interfering substances is a feature of such binding. Preferably, the binder, antibody or antibody fragment that specifically binds the target binds to the target with high affinity rather than to non-target substances. Preferably, the binder, antibody or antibody fragment that specifically recognizes or binds the target avoids binding to a substantial amount of non-target substance, e.g., non-target substance is present in the test sample. In some embodiments, the binder, antibody or antibody fragment of the invention prevents binding of more than about 90% of the non-target agent, and significantly to higher ratios. For example, a binder, antibody or antibody fragment of the invention avoids binding to about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% and about 99% or more of non-target material. In other embodiments, the binder, antibody or antibody fragment of the invention avoids binding of greater than about 10%, 20%, 30%, 40%, 50%, 60% or 70%, or greater than about 75%, or greater than about 80% or greater than about 85% of the non-target substance.

The term "antibody" as used herein includes whole immunoglobulins or antibodies or any functional fragment of an immunoglobulin capable of specifically binding an antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, or small molecule, etc., through at least one antigen recognition site located in a variable region of the immunoglobulin, and may include any type of immunoglobulin, including, for example, IgG, IgM, IgA, IgD and IgE, IgY, which is the predominant antibody type of an avian species, such as chicken.

As used herein, "fragments thereof," "regions thereof," and "portions thereof" refer to fragments, regions, and portions that substantially retain at least one function of the full-length polypeptide.

The term "antigen" as used herein refers to a target molecule that specifically binds to an antibody through an antigen recognition site. The antigen may be monovalent or multivalent, i.e., it may have one or more epitopes recognized by one or more antibodies. Such antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, or small molecules, and the like.

The term "epitope" as used herein refers to a peptide sequence of at least about 3 to 5, preferably 5 to 10 or 15, not more than 1000 (or any integer therebetween) amino acids that defines a sequence, by itself or as part of a larger sequence, that binds to an antibody produced by the peptide sequence. The length of the fragment is not critical, for example, it may comprise almost the entire length of the antigen sequence, or even a fusion protein comprising two or more epitopes from a target antigen. The epitope used in the present invention is not limited to the exact sequence of the portion of the parent protein from which it is derived, but also encompasses sequences identical to the parent sequence, as well as modifications, such as deletions, additions and substitutions (conservative) of the parent sequence.

The terms "complementary" and "substantially complementary" may include hybridization or base pairing or formation of a double strand between a nucleotide and a nucleic acid, for example, between the two strands of a double-stranded DNA molecule and between an oligonucleotide primer and a primer binding site on a single-stranded nucleotide. Generally, complementary nucleic acids are A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand preferentially match the appropriate nucleotides, insert the nucleotides, or delete, pairing with at least about 80% of the other strands, typically matching at least about 90% to about 90%, and even about 98% to 100%. In one aspect, two complementary nucleotide sequences are capable of hybridizing, preferably less than 25%, more preferably less than 15%, even more preferably less than 5% of mismatches occur between opposing nucleotides, and most preferably no mismatches occur. Preferably, the two nucleic acid molecules hybridize under conditions of high stringency.

As used herein, "hybridization" refers to the process by which two single-stranded polynucleotides are non-covalently associated to form a stable double-stranded polynucleotide. In one aspect, the double-stranded polynucleotide thus produced may be "hybridized" or "doubled". Hybridization conditions "generally include salt concentrations of less than about 1M, usually less than 500mM, and may be less than 200 mM. "hybridization buffer" includes a buffered saline solution, such as 5% SSPE, or other buffers known in the art. The hybridization temperature may be as low as 5 ℃ but is typically above 22 ℃, more typically above 30 ℃ and typically above 37 ℃. The hybridization process typically occurs under stringent conditions, i.e., conditions under which a sequence hybridizes to a target sequence and not to other non-complementary sequences. Stringent conditions are sequence dependent and will be different in different circumstances. For example, specific hybridization of longer fragments may need to be performed at higher hybridization temperatures than for shorter fragments. The combination of parameters is more important than the absolute value of any one of the individual parameters alone, as other factors may affect the stringency of hybridization, including base composition and length of the complementary strand, presence of organic solvents, and degree of base mismatching. The usual stringency conditions are selected such that T for a particular sequence is at a defined ionic strength and pH_mA temperature of about 5c lower. Melting temperature T_mMay be the temperature at which a large number of double-stranded nucleic acid molecules are generally broken down into single-stranded nucleotide molecules. There are several calculations of T for nucleic acids in the art_mAre well known. T as indicated in the Standard reference_mA simple method of estimating the value can be represented by the equation T_m81.5+0.41 (% G + C), when nucleic acids are in 1M aqueous sodium chloride (see Anderson and Young, Quantitative Filter Hybridization, Innucleic acid Hybridization (1985)), other references (e.g., Allawi and Santa L uca, Jr., Biochemistry, 36:10581-94(1997)) include alternative methods of estimation, which calculate T_mStructural, environmental and sequence features are considered.

In general, the stability of the hybrids is a result of the action of ionic concentration and temperature.typically, hybridization reactions are carried out under conditions of low stringency, but are followed by various washes of higher stringency.typical stringency conditions include conditions of pH 7.0 to 8.3 at least 25 ℃ at least 0.01M to a salt concentration not exceeding 1M sodium ion concentration (or other salts). for example, 5 × SSPE (750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH 7.4) and a temperature of about 30 ℃ suitable for allele-specific hybridization, although the appropriate temperature depends on the length and/or GC content of the hybridization region. in one aspect, "hybridization stringency" which determines the percent mismatch is as follows: 1) high stringency: 0.1 × SSPE, 0.1% SDS, 65 ℃; 2) stringency: 0.2 × 0SSPE, 0.1% SDS, 50%, also called appropri.2% SDS, 0.×% SDS, 50% SDS, 65 ℃; 2, 2% SDS, 1% SDS, 35% SDS, 10% SDS, 5% SDS, or 5% SDS, 5% SDS, 5% or 5% SDS, similar to the conditions of a high stringency, such as a high stringency, medium of a buffer (such as a buffer, 5% SDS, 5% SDS, 5% sodium chloride, 5% SDS, 5% sodium alginate, 5% sodium, 5% sodium chloride, 5% sodium, 5% and 5% sodium, 5% SDS, 5% sodium alginate, 5% sodium alginate, 5% SDS, 5% SDS, 5% SDS, 5.

Alternatively, substantial complementarity exists when an RNA or DNA strand hybridizes to its complementary strand under selective hybridization conditions. Typically, selective hybridization will be transmitted when there is at least 65% complementarity on a strand having at least 14 to 25 nucleotide molecules, preferably at least 75%, more preferably at least 90% complementarity. See, for example, M.Kanehisa, Nucleic Acids Res.12:203 (1984).

As used herein, a "primer" can be a natural or synthetic oligonucleotide that is capable of forming a duplex with a polynucleotide template, of serving as a point of initiation of nucleic acid synthesis, and of extending from its 3' terminus along the template, thus forming an extended duplex. The polynucleotide sequence added during the extension process is determined by the polynucleotide template sequence. The primer is typically extended by a DNA polymerase.

"ligation" refers to the formation of covalent bonds in a template-driven reaction or the formation of a ligation of two or more nucleic acid molecule ends, such as an oligonucleotide and/or polynucleotide. The nature of the covalent bond or linkage may vary widely and the linkage may be effected enzymatically or chemically. As used herein, ligation is generally under the action of an enzyme to form a phosphodiester linkage between the 5 'carbon terminal nucleotide of one oligonucleotide and the 3' carbon terminal nucleotide of another nucleotide.

"high throughput digital sequencing" or "next generation sequencing" refers to sequence detection that detects many (typically thousands to billions) of nucleic acid sequences in an intrinsically parallel manner, i.e., sequencing of a DNA template is not performed one at a time, but rather is a batch-wise process, and preferably many sequences are read in parallel, or an ultra-high throughput serial procedure in parallel with itself^TMtechnology, L if Technologies, Inc., Carlsbad, Calif.), sequencing using modified nucleotide synthesis (e.g., commercially available TruSeq)^TMand HiSeq^TMtechnology byIllumina，Inc.，San Diego，Calif.；HeliScope^TMby Helicos Biosciences Corporation, Cambridge, Ma. and PacBio RS by Pacific Biosciences of California, Inc., Menlo park, Calif.), sequencing by Ion detection techniques (e.g., Ion Torrent^TMtechnology, L if Technologies, Carlsbad, Calif.), DNA nanosphere sequencing (Complete Genomics, Inc., Mountain View, Calif.), Nanopore sequencing Technologies (e.g., developed by Oxford Nanopore Technologies, L TD, Oxford, UK), methods such as highly parallel sequencing methods.

"SNP" or "single nucleotide polymorphism" includes genetic variation between individuals; for example, a single nitrogenous base position of the variable organism DNA. Single nucleotide polymorphisms exist throughout the genome; genetic variation among individuals is due to variation in SNP sites, and often this genetic variation results in phenotypic variation among individuals. The SNPs and respective alleles used in the present invention may be from any number of data sources, such as public databases (genome. ucsc. edu/cgi-bin/hgGateway) or NCBI dbSNP website (www.ncbi.nlm.nih gov/SNP /), or may be experimental assays such as those described in U.S. publication nos. 6969589 and 2006/0188875 entitled "human genome polymorphisms," although in the examples herein, it is understood that genetic markers for other bi-or multi-alleles may also be used. For each biallelic polymorphism (e.g., disease or drug response) associated with a given trait, there is a corresponding associated allele. Other biallelic polymorphisms used in the methods provided herein include, but are not limited to, alteration, insertion, deletion, and translocation of a polynucleotide. Further, reference herein to DNA may include genomic DNA, mitochondrial DNA, episomal DNA, and/or DNA derivatives such as amplicons, RNA transcripts, cDNA, DNA analogs, and the like. The polymorphic sites screened for in association studies may be diploid or haploid chromosomal in state, ideally from sites on the genome.

As used herein, the term "microfluidic device" generally refers to a device through which materials, particularly fluid materials such as liquids, may be transported, in some embodiments on the microscale, and in some embodiments on the nanoscale. Accordingly, the microfluidic devices described herein may contain microscale features, nanoscale features, and combinations thereof.

A typical microfluidic device, therefore, generally includes structural or functional features that are measured on the order of millimeters or less, which device is capable of manipulating fluids at flow rates of μ L/minute or less.

A typical microfluidic device, therefore, generally includes structural or functional features that are measured on the order of millimeters or less, which device is capable of manipulating fluids at flow rates of μ L/minute or less.

As used herein, the terms "channel", "microchannel", "liquid channel" and "microfluidic channel" are used interchangeably and refer to a recess or cavity formed in a material by a demolding technique that introduces the pattern of a patterned substrate onto the material or any suitable material, or may refer to a recess or cavity, such as a tube, capillary or the like, in combination with a liquid directing structure mounted in the recess or cavity. In the present invention, the channel size means the cross-sectional area of the microfluidic channel.

As used herein, the terms "liquid channel" and "control channel" are used interchangeably and may refer to a channel in a microfluidic device through which a material, such as a liquid, such as a gas or liquid, may flow. In particular, the term "liquid channel" refers to a channel through which a material of interest, such as a solvent or chemical agent, can flow. Furthermore, the term "control channel" refers to a flow channel through which a material, such as a liquid, for example a gas or a liquid, can flow and thus open a valve or a pump.

As used herein, a "chip" may refer to a solid substrate having a plurality of one-dimensional, two-dimensional or three-dimensional microstructures or micro-scale structures upon which certain processes, such as physical, chemical, biological, biophysical or biochemical processes, and the like, may be performed. Microstructure or micro-scale junctionsStructures such as channels and wells, electrode elements, electromagnetic elements are incorporated, constructed or attached to the substrate to facilitate physical, biophysical, biological, biochemical and chemical reactions or processes occurring on the chip. The chip may be thin in one dimension and may have various shapes in other dimensions, for example, rectangular, circular, oval or other irregular shapes. The dimensions of the major surfaces of the chips of the invention may vary widely, for example from about 1mm²To about 0.25m². Preferably, the size of the chip is from about 4mm²To about 25cm²Having a gauge size from 1mm to 5 cm. The chip surface may be flat, or non-flat. A chip with a non-flat surface may include vias or holes configured in the surface.

Microfluidic chips may be used in the methods and analytical systems disclosed herein. The microfluidic chip may be made of any suitable material, such as PDMS (polydimethylsiloxane), glass, PMMA (polymethyl methacrylate), PET (polyethylene terephthalate), PC (polycarbonate), etc. or a combination thereof.

Microfluidic chips may be used in the methods and analytical systems disclosed herein. The microfluidic chip may be made of any suitable material, such as PDMS (polydimethylsiloxane), glass, PMMA (polymethyl methacrylate), PET (polyethylene terephthalate), PC (polycarbonate), etc. or a combination thereof.

Assays for detecting spatial distribution of biological targets

The invention discloses a spatial coding, and discloses a multiplex analysis method and an analysis system for high-level multiplex analysis by adopting an effective spatial coding scheme. In one embodiment, a test device is provided that is capable of delivering a reagent to a sample, thereby spatially encoding multiple sites at which the reagent is delivered. In one aspect, the reagent may be delivered to the sample according to a known spatial distribution, e.g., determined by the tissue properties of the sample. Microfluidic devices and the like having addressing channels, on the other hand, are used to provide reagents to a sample and spatially encode multiple sites in the sample at which reagents are delivered. In some embodiments, the spatially encoded ("tag" or "tag label"), multiplex analysis methods and analysis systems include a decoding feature determined by a digitized reader. In one aspect, the disclosed methods and assay systems detect the presence or absence of a biological target or an indicator of biological activity of a biological target. In another aspect, the invention provides methods and assay systems that can detect the amount or abundance of a biological target or an indicator of the activity of a biological target at a plurality of sites in a sample, as well as the location of each of the plurality of sites in the sample. Based on the analysis of the quantity or abundance and the analysis and activity of the positional information of one or more biological targets, a spatial distribution of multiple sites in the sample can be generated. In any of the preceding embodiments, the method or analysis system may not rely on imaging techniques to detect spatial or positional information of one or more biological targets in a sample, although the method and system may optionally include using the imaging techniques for other purposes. Imaging techniques may include, but are not limited to, conventional Immunohistochemical (IHC) imaging and Immunofluorescence (IF) imaging techniques. Methods and analytical systems for detecting the spatial distribution of abundance and/or activity of biological targets in a sample are disclosed in detail in U.S. application Serial No.13/080,616, entitled "spatial encoded biological assays" (pub.no.: US 2011/0245111), the disclosure of which is incorporated herein in its entirety for the purposes of reference.

The invention further provides a device capable of delivering a reagent to a plurality of sites in a sample, wherein each site is capable of being recognized by the delivered reagent. In one embodiment, the agent is delivered in a spatially distributed manner. The apparatus and software, reagents and protocols for this experiment provide key components of the methods and assay systems of the invention, allowing the detection of a large number of biological targets or activities, including DNA, RNA and/or protein expression and spatial localization of biological targets and activities in a sample. In one embodiment, after the products of the multiplex assay are removed from the biological sample and combined for analysis, the abundance, expression and/or activity and localization of the biological target of the biological sample is detected. Detection of abundance, expression, and/or activity and localization of biological targets can be achieved by, for example, next generation sequencing, which can easily provide millions and trillions of data at low cost. The results of the analysis, such as the amount or activity of the biological target, can be mapped back to the specified location of the biological sample. Such methods and analysis systems provide tools for analyzing complex cellular functions and regular spatial distributions in biological samples.

In one aspect, a method for detecting the spatial distribution of abundance, expression and/or activity of one or more biological targets at a plurality of sites in a sample is shown in figure 1. At step 110, a probe for each of one or more biological targets, each probe comprising a target binding group, an address tag identifying the probe delivered to each site, and an identification tag, is delivered to a plurality of sites in a sample.

In any of the preceding embodiments of the invention, the sample may be any biological sample or sample that may be attached to a support or provided in a two-dimensional manner such that the biological target or activity being analyzed may be bound to a location in the biological sample. In certain embodiments, the sample can be a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a treated sample, or a combination thereof. Exemplary samples of the invention include tissue sections (e.g., including whole animal sections and tissue biopsies), cell populations, or biological structures processed on other supports, such as slides (e.g., microscope slides) or petri dishes, among others. In preferred embodiments, the methods and analytical systems of the present invention are compatible with a wide variety of biological sample types, including fresh samples, such as primary tissue sections and preserved samples including, but not limited to, frozen samples and formaldehyde-fixed samples, paraffin-fixed (FFPE) samples. In certain embodiments, the sample can be fixed with a suitable concentration of formaldehyde or paraformaldehyde, for example, 4% formaldehyde or paraformaldehyde in Phosphate Buffered Saline (PBS). In certain embodiments, the biological sample is immobilized on a substrate surface having separate, independent measurement areas.

In one embodiment, the biological sample may comprise one or more biological targets of interest. In any of the preceding embodiments of the invention, the one or more biological targets may be any biological molecule including, but not limited to, a protein, a nucleic acid, a lipid, a carbohydrate, an ion, or a complex comprising multiple components of any of the foregoing molecules. Examples of subcellular targets include organelles, e.g., mitochondria, golgi apparatus, endoplasmic reticulum, chloroplasts, endocytosis vesicles, vacuoles, lysosomes, and the like. In some embodiments, the one or more biological targets can be nucleic acids, including RNA transcripts, genomic DNA sequences, complementary DNA, amplicons, or other nucleic acid sequences. In other embodiments, the one or more biological targets can be proteins, enzymes (proteases or ribozymes), and the like.

At step 110, the probe for each of the plurality of biological targets comprises: (1) a target binding group capable of binding to a corresponding biological target of the probe; (2) an address tag identifying each site to which the probe is delivered; and (3) an identification tag that identifies the corresponding biological target or target binding group of the probe. Depending on the nature of the biological target, the target binding group may be a target-specific nucleotide sequence (e.g., the complement of a nucleic acid target sequence), a small molecule, a nucleic acid aptamer, an antibody, a lipid, a carbohydrate, an ion, an affinity capture agent, or a multiplex complex comprising any of the above molecules. The location tag identifies the location in the sample to which the probe is delivered, and the identification tag identifies the corresponding biological target or target binding group being analyzed. Thus, the identification of the address tag and the identification tag can be used to correlate the results of the analysis with the biological target and location in the sample. In a preferred embodiment, there are at least two address tags of the biological target at each of the plurality of sites in the sample, each address tag identifying a parameter at each of the plurality of sites. For example, an X abscissa address label and a Y ordinate address label on an X-Y coordinate plane represent each site in the sample. Thus, each site can be uniquely identified by its corresponding (X, Y) plane coordinates. In a preferred embodiment of the invention, the address tag and/or the identification tag may be an oligonucleotide. In other embodiments, the address tag and/or identification tag may be a large-scale tag, a fluorescent tag, or other group.

In some embodiments, the target binding moiety, address tag, and/or probe identification tag are pre-paired prior to delivery to the biological sample. In the case where the probe is an oligonucleotide, the target binding sequence, the address tag sequence, and/or the identification tag sequence may be synthesized as a single oligonucleotide. Alternatively, the target binding moiety, address tag, and/or the identification tag of the probe may be synthesized or obtained separately prior to delivery to the sample and bound prior to being provided to the biological sample. For example, two separate oligonucleotides may be prepared and combined separately, e.g., by ligation, prior to delivery to the sample. Alternatively, the antibody and oligonucleotide may be prepared separately and combined prior to delivery to the biological sample. In other embodiments, the probe and address tag can be synthesized separately and delivered to the biological sample at different steps of the assay (e.g., probe-first, address tag-immediately-thereafter; or vice versa).

In step 120, the probes may bind to the corresponding biological targets in the sample, thereby reacting or interacting with the biological targets. For example, conditions are provided that allow for hybridization of the oligonucleotide to the nucleic acid target, catalytic reaction of the enzyme with the protein target, binding of the antibody to an epitope in the target, and the like. Where the biological target is a nucleic acid, the probe is typically an oligonucleotide and hybridizes to the target nucleic acid. In the case where the biological target is a protein, the probe is an oligonucleotide aptamer, a small molecule or an oligonucleotide-coupled protein that binds to or reacts with the target protein (i.e., where one protein is a substrate for another protein). The oligonucleotide may be bound to the probe or protein by linkage of suitable groups and the like, chemical or photocrosslinking reaction.

In some embodiments, after allowing the probes to bind or interact with one or more biological targets in the sample, the probes bound to the biological targets can be separated from the probes delivered to the sample without binding to the biological targets. In one aspect, where the biological target is a nucleic acid and the probes are oligonucleotides, the separation is achieved by, for example, eluting non-hybridized probes from the sample, and similarly for other assays based on affinity binding, including the use of aptamers, small molecules and protein probes, a washing step may be used to remove low affinity binders. In the case of probes that are converted by interaction with the target, e.g., in the case of targets that are peptides, such as by cleavage by proteases or phosphorylation by kinases, it is convenient to collect all probes, including probes that have interacted with the biological target and thus converted probes and unconverted probes. After pooling or pooling, antibodies or other affinity capture agents can be used to capture the probes after conversion by increasing groups (e.g., phosphate groups phosphorylated by kinases). In the case where the probes are converted by cleavage, the converted probes may be separated, for example during the conversion (e.g. by cleavage), by capturing the non-converted probes via a removed label on the converted probes, or by adding a new label at the site of cleavage.

In some embodiments, the analysis comprises detecting the abundance, expression, and/or activity of each biological target and analyzing the identity of the identification tag and address tag of each target at each site^TML C/MS/MS), magnetic resonance imaging technology, or, preferably, nucleic acid sequencing technology to detect address tags, for example, an example of the probe decoding technology of the present invention can be found in U.S. publication No.20080220434, which is hereby incorporated by reference in its entiretyOne amplification is specific for a particular address tag or subset of tags, and amplification is accomplished by using tag-specific primers. Each amplification also incorporates a different soluble tag (e.g., fluorophore). After hybridization, the relative amount of a particular target mapped to different spatial locations in the sample can be determined by the relative abundance of the soluble tags. In step 140, the abundance, spatial distribution of expression and/or activity of one or more biological targets at a plurality of sites in the sample is detected based on the analysis of probes bound to the one or more biological targets.

In a preferred aspect, probes according to the invention are substrates for high throughput and next generation sequencing, and highly parallel next generation sequencing methods are used to determine the sequence of the probes (e.g., sequences comprising target binding groups, address tags, and/or recognition tags)^TMTechniques (Life technologies Inc.) or genomic analyzers (Illumina Inc.) such next generation sequencing methods can Be used, for example, by using single-pass sequencing methods or end-pair sequencing methods including, but not limited to, hybridization-based methods such as those disclosed in Drmanac, U.S. Pat. Nos.6, 864, 052, 6, 309, 824 and 6, 401, 267, and Drmanac et al, U.S. patent publication 2005/0191656, synthetic sequencing methods such as U.S. Pub.Nos.6,210,891, 6,828,100, 636,969,488, 6,897,023, 6,833,246, 6,911,345, 6,787,308, 7,297,518, 7,462,449 and 7,501,245, U.S. application Ser. No. 20110059436, 20040106110, 20030064398 and 20030022207, Ronaghi, et al, Science, 281:363-365 (and L i, et al, Proc.Natl., Acad.Sci., 100: 19834, and EP, 19835; US patent publication No. 2005926,482, 4354, and US patent publication No. 20046,52; US patent publication No. 2005926,220,220,220,482, US patent publication No. 3,52; US patent publication No. 3,201,8652; and US patent publication No. 3,201,500,500,150; US patent publication No. 3,52; US patent publication No. 3,500,500,150; US patent publication No. 3,150,150; US patent publication No. 3,500,150; US patent publication No. 3,150; US patent No. 3,150,150,52; No. 3,150,150; No. 3,52; No. 4,52; No. 3,150; Nontley, et al, Nature 456:53-59(2008), all of which are incorporated herein by reference in their entirety.

In a preferred embodiment, probes that bind to one or more biological targets are analyzed by sequencing. Such analysis by sequencing includes determination of the number of sequencing products, which may indicate abundance, expression, and/or activity of each biological target, the sequencing products comprising all or part of the address tag sequence and all or part of the recognition tag sequence that recognizes each biological target at each site. In one embodiment, the address tag sequence of the sequencing product allows the test results to be mapped back to the multiple sites in the sample.

In certain embodiments, two probes that bind to the same target molecule (e.g., two polynucleotide probes that hybridize at adjacent sites of a nucleic acid target) can be analyzed by post-extension ligation (the extension-ligation assay). The extension-ligation assay allows for the re-detection of specific target sequences. For example, if the primer and downstream oligonucleotide are separated by 20 bases and a reverse transcriptase is used to fill the 20 base gap, 20 bases of the RNA target sequence can be acquired. In certain embodiments, a particular sequence region of interest may be characterized by extension-ligation analysis using the methods or analysis systems of the invention. For example, these regions may contain mutations or variations, e.g., affecting cancer, MHC differences and RNA editing.

In any embodiment of the invention, extension assays may be used and allow certain target sequences to be re-detected. In one embodiment, the extended assay of the invention is performed as follows: first, a first primer is used to synthesize cDNA from a target sequence. In certain embodiments, the primer can be a random primer (e.g., a random hexamer) or a sequence specific primer. Random primers can be used to make cDNA from the entire transcriptome, while sequence-specific primers are used to make cDNA from specific target sequences. In certain aspects, the first primer may comprise a universal primer site for analysis of product amplification, an adaptor for sequence recognition by sequencing techniques, and/or an adaptor for attaching an address tag. The first primer is attached to a linker for attaching an address tag. The X and Y address tags described below are then bound to the first primer via the linker. Note that in this case, the X and Y address tags are bound to the same side or different sides of the target sequence, and this configuration can be used in any embodiment of the invention. The Y address tag in this case is further attached to a universal primer site or biotin-coupled linker for sequencing. The cDNA with the bound X and Y address tags is then eluted and captured on streptavidin magnetic beads, and then a second primer is installed on the cDNA at the opposite site of the first primer. In some embodiments, capture of the polynucleotide sequence may be based on other hapten binders, or on the sequence rather than biotin-streptavidin. In certain embodiments, the second primer can be a random primer (e.g., a random hexamer) or a sequence specific primer. In certain aspects, the primers may comprise universal primer sites for analysis of product amplification, adapters for recognition of sequences by sequencing techniques, and/or adapters for attachment of address tags. In other embodiments, the second primer may bind to a universal primer site or to a linker for sequencing. Coupled to biotin with a universal primer site or linker, a sequence can be extended, amplified and sequenced from the second primer.

The method of the invention the analysis system may comprise an amplification step, in particular a nucleic acid amplification step. In certain aspects, the amplifying step is performed by PCR. In certain embodiments, linear amplification (by T7RNA polymerase) may be used in place of or in part of PCR. In some aspects, linear amplification of polynucleotides may cause less distortion in the relative abundance of different sequences. This can be accomplished by including the T7RNA promoter in a common portion of the sequence. In this case, the promoter itself and the upstream portion of the promoter are not replicated. In still other embodiments, other amplification methods may be used in the methods and assay systems of the invention. For some sequencing methods (e.g., nanopore sequencing), amplification is optional.

T7RNA polymerase-based amplification is a routine procedure for mRNA amplification, originally described in Proc.Natl.Acad.Sci.USA 87 by van Gelder et al,1663, 1667 (1990). The procedure involves the synthesis of cDNA complementary to mRNA ("first strand synthesis"), which is produced by reverse transcription followed by second strand synthesis to produce double-stranded cDNA, and in vitro transcription which is initiated by T7RNA polymerase using the double-stranded cDNA as a template. The last step provides single stranded antisense rna (arna), which can be labeled when labeled nucleotides are provided. The nucleotides may be labelled by radiolabelling or non-radiolabelling means. The method of van Gelder was extended by a second round of amplification by addition of RNA obtained in the first round as template by Eberwine et al (Proc. Natl. Acad. Sci. USA 89, 3010-3014 (1992)). Wang et al (NatureBiotechnol.18, 457-. Second chain Synthesis method SMART of Wang et al^TMThe technique (Clontech) is a method known in the art for cDNA synthesis. Baugh et al (Nucleic Acids Res.29, E29(2001)) described optimized variations and analyzed Affymetrix GeneChips according to van Gelder et alThe performance of (c). The Affymetrix gene chip is designed to detect the antisense strand. When performing T7RNA amplification, any other DNA chip or microarray that probes the antisense strand is envisioned, wherein labeling occurs during the in vitro transcription step.

In other embodiments, amplification techniques such as Rolling Circle Amplification (RCA) and loop-to-loop amplification (C2CA) may be used for the probes, targets, tags, and/or signal amplification of the invention. RCA is a linear isotherm process in the presence of certain DNA polymerases using ssDNA minicycles as templates (Fire and Xu, Proc. Natl. Acad. Sci., 92: 4641-78145 (1995); Daubendek et al., J.Am. chem. Soc.117:77818-7819 (1995)). In certain aspects, polynucleotide sequences may be amplified 500 to 1000 fold depending on the amplification time. For example, in a dual target assay for a target protein as discussed above, linear ligation products are formed (e.g., when two antibodies bind to adjacent regions of the target protein, the oligonucleotide tags of the antibodies can be ligated) and can be cleaved by restriction enzymes and then ligated by DNAThe enzyme and template are re-ligated to form a DNA loop. In certain embodiments, phi29 DNA polymerase can be used to extend a primer, which is also templated, to form a long ssDNA comprising a large number of sequences complementary to the original DNA circle. C2CA is based on RCA and may include three steps: replication, monomerization and ligation (Dahl et al, Proc. Natl. Acad. Sci., 101: 4548-. The original circular DNA is considered to be positive. After a replication step (RCA reaction), the product is switched to the opposite polarity. With positive polarity (RO)⁺) The restriction oligonucleotide of (a) may form a diploid region with the RCA product, which diploid region may be cleaved by the restriction enzyme to produce a monomer. The monomer is then directed to a ligation step and cyclized. These loops serve as templates for the next round of RCA, as defined by RO⁺And (6) guiding. This method can be further repeated to produce target sequences at concentrations of about 100-fold or more than conventional PCR.

In another aspect, as shown in fig. 2, a method for detecting the spatial distribution of abundance, expression, and/or activity of one or more biological targets at a plurality of sites in a sample is provided, describing the efficient implementation of an address tagging scheme for one or more biological targets at a plurality of sites. In one embodiment, the address tag protocol is a combination protocol using at least two address tags for a plurality of biological targets at each of a plurality of sites in the sample. In step 210, a probe for each of a plurality of biological targets at a plurality of sites in a sample is delivered, each probe comprising (1) a target binding moiety capable of binding to the probe's corresponding biological target; (2) and (3) identifying a label, and identifying a corresponding biological target or target binding group of the probe. Depending on the nature of the biological target, the target binding moiety may be a target-specific nucleotide sequence (e.g., a sequence complementary to a nucleic acid target sequence), a small molecule, a nucleic acid aptamer, an antibody, a lipid, a carbohydrate, an ion, an affinity capture agent, or a multicomponent complex comprising any of the above molecules. In step 220, each probe is allowed to interact or bind with a corresponding biological target in the sample under suitable conditions.

In certain embodiments, probes that are not bound to a biological target may be removed to separate from those bound to the biological target. Such isolation is performed substantially as discussed above, e.g., by washing the sample to remove unhybridized oligonucleotide probes. In certain embodiments, to detect the spatial distribution of abundance and/or activity of a biological target, probes bound to the biological target may not have to be separated from probes not bound to the biological target.

Next, in step 230, the address tag is delivered to each of a plurality of sites in the sample, and the address tag is paired with the probe of each biological target and identifies each site to which an address tag is delivered. Note in this regard that the probe and the address tag are delivered in separate steps. In certain embodiments, when the probe is an oligonucleotide, the address tag can be bound to the oligonucleotide probe in a variety of ways known to those skilled in the art, for example, by extension, ligation after extension, or any combination thereof. For example, the information of the address tag is transferred by means of extension of a probe oligonucleotide as a primer by amplification with a DNA polymerase, thereby copying and merging the sequence of the address tag.

In step 240, the probe/address tag conjugates bound to one or more biological targets are analyzed. In certain embodiments, the analysis comprises detecting the abundance and/or activity of each biological target and identifying the identification tag and address tag of each biological target at each site. As discussed above, a number of methods are used to identify the address tag, the target binding group of the tag and/or probe. In a preferred embodiment, probe/address tag conjugates that bind to one or more biological targets are analyzed by sequencing. Any suitable sequencing techniques and methods as discussed above may be used, including high throughput, next generation sequencing and highly parallel next generation sequencing methods. Preferably, in any one of the preceding embodiments of the invention, all or part of the address tag sequence and all or part of the identification tag sequence are determined from the same sequence product. Preferably, the abundance and/or activity of the sequence products is determined simultaneously, which is indicative of the abundance and/or activity of each biological target. In some embodiments, the abundance of the sequence product reveals the relative amount of biological target at the site.

Based on the analysis of probe/address tag conjugates bound to one or more biological targets in step 240, a spatial distribution of abundance and/or activity of one or more biological targets at a plurality of sites in the sample is detected in step 250, for example, by plotting the abundance and/or activity of each biological target tested to each site in the sample.

Although in certain embodiments the various steps are discussed in a particular order to better explain the subject matter of the present invention, the exact order of the steps may be different. For example, steps 210 and 230 may be combined so that a mixture of probes and address labels may be delivered. The binding of the address tag to the probe may occur immediately after or simultaneously with the combining of steps 210 and 230. It will therefore be appreciated that the separation of the address tag of the probe molecule from the probe based on the ability of the probe to interact with its corresponding target can be achieved flexibly. Similarly, the address tag scheme also has considerable flexibility. As will be described below, the disclosed methods and analytical systems are particularly suited for combinatorial approaches.

Spatially encoded genomic analysis

In particular embodiments, the methods and assay systems can be used for nucleic acid analysis, e.g., for genotyping, detecting Single Nucleotide Polymorphisms (SNPs), quantifying the amount of DNA replication or RNA transcription, specific transcriptional localization in a sample, and the like. Figure 3 shows a typical assay and address tagging scheme. For purposes of illustration, where the target is a nucleic acid sequence, two oligonucleotide probes are provided, it being understood that the disclosed methods and assay systems may be used with any suitable target utilizing one or more suitable probes. Each oligonucleotide probe includes a target-specific region, see 305 and 307, respectively. In certain embodiments, e.g., detection of SNPs, two target-specific regions are located on either side of the SNP to be analyzed. Each oligonucleotide probe also includes a linker region, see 301 and 303, respectively. The oligonucleotide probe is allowed to hybridize to a target nucleic acid (not shown) in the biological sample. In step 302, one or both of the oligonucleotide probes may be extended and ligated to other probes to form an extension probe comprising 309 the target nucleic acid region and the ligation regions 301 and 303. In some embodiments, two probes are in close proximity to each other and need only be connected to form one extension probe. In some embodiments, step 302 is used to merge one SNP sequence or other target sequence to be analyzed.

In step 304, two address tags, both of which comprise an address tag region (see details 315 and 317), a ligation region (see details 311 and 313), and a primer region (see details 319 and 321), are ligated to the extended probe to form a target-specific oligonucleotide. In contrast to fig. 1, the probe and address tag are delivered separately. In some embodiments, a pair of address tags are specifically attached to one or the other side of the target sequence (e.g., the 5 'and 3' ends of the target sequence), respectively. In certain embodiments, the linker region and primer region of the address tag and the probes are universal, i.e., the arrangement of linker region and primer region used to construct the probes and address tag is consistent, differing only in the target-specific region of the probes and the address tag region of the address tag. In further embodiments, the ligation region and primer region are generic, and each probe and/or address tag may comprise a different location region and/or primer region.

After ligation, probe/address tag conjugates are eluted, pooled, and optionally, sequenced linkers are added to the probe/address tag conjugates by PCR. In further embodiments, the sequencing primer may be attached to the address tag, or the sequencing primer sequence may be included in the address tag as part of the address tag. As shown in fig. 3, each sequencing adapter comprises primer region 319 or 321 and is compatible with primer regions 319 and 321 of the address tag. The final structure comprising the first said linker 327, the first primer region 319, the first coding tag 315, the ligation regions 311 and 301, the target region 309, the ligation regions 313 and 303, the second coding tag 317, the second primer region 325 and the second linker 329 is subjected to sequencing, for example by entering a digital high throughput sequencing program.

The combination of extension and ligation reactions is illustrated in FIG. 3 and includes only ligations (e.g., oligonucleotides that hybridize to contiguous portions of the target nucleic acid sequence), but it should be understood that a variety of reactions may be used to bind the address tag to the target-specific probe. Alternatively, assays using additional oligonucleotides may be employed, e.g.Analysis, (Illumina, Inc., San Diego, Calif.) (see Fan, et al, Cold Spring Symp. Quant. biol., 68:69-78(2003)) for details.

To maximize the efficiency of address tagging, a joint approach using a pair of address tags may be taken. By combining tag-specific information with spatial information in the address tag, the number of spatially distributed oligonucleotides used to detect one or more biological targets at multiple sites in a sample can be significantly reduced, with concomitant reduction in cost.

FIG. 4 illustrates one embodiment of a combined address tagging scheme in which nucleic acids in representative tissue sections are analyzed (as shown at 416). Fig. 4A shows that two probe/address tag conjugates 420 and 422 specifically bind to the biological target of interest 402. The first probe/address tag conjugate 420 comprises an address tag 408 associated with the tag 404. The tag 404 may be a universal primer site for analyzing the amplification of the product or an adaptor that recognizes other regions of the address tag 408 and/or probe/address tag conjugate 420, for example, using sequencing techniques. The second probe/address label conjugate 422 comprises an address label 406 associated with the label 410. The tag 410 may be a universal primer site for analysis of product amplification or an adaptor that recognizes the address tag 406 and/or other regions of the probe/address tag conjugate 422, for example, using sequencing techniques.

In other embodiments, as shown in fig. 4D, the biological target 424 is analyzed according to a combined address tagging scheme. Two probes 426 and 428 specifically bind to the biological target of interest 424. In some embodiments, for example, a portion of each probe of probes 426 and 428 specifically binds to a target, and a portion of each probe also specifically binds to the linker 438, e.g., by specific nucleic acid hybridization. In one embodiment, the probe or probes specifically hybridize to the linker. Where the biological target is a nucleic acid and the probe is an oligonucleotide, the linker may specifically bind to the following composition: 1) a 5 'portion of probe 426 and a 3' portion of probe 428; 2) a3 'portion of probe 426 and a 5' portion of probe 428; 3) a 5 'portion of probe 426 and a 5' portion of probe 428; or 4)) a3 'portion of probe 426 and a 3' portion of probe 428. In certain embodiments, probes 426 and 428 are linear molecules, branched molecules, circular molecules, or combinations thereof. After the two probes bind to the biological target and the adaptor binds to the two probes, an address tag can be delivered to the sample and bind to the adaptor. For example, the joint may be labeled with address label 430, associated with label 434, and/or associated with address label 432, associated with label 436. For example, using sequencing techniques, tags 434 and 436 can be universal primer sites for analysis of product amplification or sequences that can recognize address tags and/or other regions of the linker/address tag conjugate. In certain embodiments, the address tags are labeled at the same end or different ends of the linker. In other embodiments, the address tag and/or labels 434 or 436 may be pre-bound to the linker, and the linker/address tag or linker/tag conjugate or complex then delivered to the sample to bind to the probe bound to the biological target. In certain aspects, the linker is a linear molecule, a branched molecule, a circular molecule, or a combination thereof. In some embodiments, after an address tag is attached to each end of the junction, the ends may be joined. For example, in fig. 4D, address tags 434 and/or 436 contain structure and/or sequence 1 to allow the two ends of the labeled linker 438 to be joined to form a circular configuration, which facilitates amplification and/or sequencing of the configuration.

In certain embodiments, all or a portion of the linker/address tag conjugate sequence is determined, for example, by nucleic acid sequencing techniques. In other embodiments, all or part of the probe sequence, all or part of the linker/address tag conjugate sequence is detected. For example, a first address tag can be attached to probe 426 and a second address tag can be attached to linker 438. The double complex formed between probe 426 and the linker 438 can be used for extension and sequencing, resulting in a conjugate comprising a first address tag sequence, all or part of the sequence of probe 426, all or part of the sequence of the linker 438, and a second address tag sequence.

The labeling scheme is not limited to the use of two or more probes directed to the same biological target. For example, where a probe is employed, a tag (e.g., an address tag, a ligation adapter, or a universal sequencing primer or amplification primer sequence) can be bound to the adapter that specifically binds the probe, rather than to the probe itself.

In some embodiments, at least two of the joints are used. In one aspect, more than one probe is delivered to the sample, and at least one linker is provided for each probe that specifically binds to the probe. In one aspect, one or more linkers are provided for specifically binding each probe. For example, a pair of linkers is used to specifically bind the probes 426 and 428, respectively. In certain embodiments, a pair of the linkers are DAN molecules that: 1) hybridization or otherwise binding to probes 426 and 428; 2) having free 3 'and/or 5' ends that enable coding sequences (e.g., address tags 430 and 432) to be attached in a subsequent step, e.g., by ligation; 3) in a form that can be connected if they are co-located or in close proximity to each other. In some embodiments, portions of probes 426 or 428 are cleated as joints in the joint pair so as to be able to connect or extend and connect. Additional tags (e.g., address tags, ligation adaptors, or universal sequencing primers or amplification primer sequences) can be attached to the adaptors generated by ligating the adaptor pairs.

FIG. 4B shows an address tag scheme that can be used for 100 individual sites in a sample. For example, twenty probe/address tag conjugates a1 through a10 and b1 through b10 were used, as well as each of a1 through a10 for probe/address tag conjugate 420 (containing address tag 408) and each of b1 through b10 for probe/address tag conjugate 422 (containing address tag 406). Each address tag contained in a1-a 10 and b1-b 10 is uniquely identified. The probe/address tag conjugate a1, for example as shown in the first horizontal channel in 412, is delivered to the biological sample through an addressing channel. The probe/address tag conjugate a2 is delivered to the biological sample through a second horizontal channel in 412. The probe/address tag conjugate a3 is delivered to the biological sample via a third horizontal channel in 412, and so on. As shown at 414, "a" probe/address tag conjugates are delivered in 10 horizontal channels, whereas "b" probe/address tag conjugates are delivered in 10 vertical channels. For example, probe/address tag conjugate b1 is delivered to the biological sample via a first horizontal channel in 414, probe/address tag conjugate b2 is delivered to the biological sample via a second horizontal channel in 414, and so on. In other embodiments, the "a" tag may refer to the "X" tag and the "b" tag to the "Y" tag. Each intersection or junction may be uniquely identified independently by a conjugate of an "a" probe/address tag conjugate and a "b" probe/address tag conjugate delivered to the sample region corresponding to the intersection or junction.

Fig. 4C shows a representative tissue slice 416 that is coincident with grid 418. Arrows indicate how the "a" probe/address tag conjugates and the "b" probe/address tag conjugates are delivered in grid 418 coincident with tissue section 416. For example, if, for example, probe/address tag conjugates a1 and b4 associated with the target, once analyzed, indicate that the target is present in the tissue section at location (a1, b 4).

The methods and assay systems disclosed herein are capable of multiplex assays. For example, FIG. 5 provides an address tagging (or "location coding") scheme that may be used for multiple detections. For a clear illustration, two probes TS01 and TS02 are specific for target 1 and target 2, respectively, as shown at 520. FIG. 5 shows address tag 510, which includes a1, a2, a3, a4 and b1, b2, b3 and b 4. The delivery or allocation scheme is shown at 530. As shown in the grid of FIG. 4, a1-a4 is delivered to the sample through a horizontal channel and b1-b4 is delivered to the sample through a vertical channel. The direct intersection of the horizontal and vertical channels is shown as a solid square of a cube. Each intersection or junction may be uniquely identified by a conjugate of an "a" probe/address tag conjugate and a "b" probe/address tag conjugate in the sample region delivered to the corresponding intersection.

Probes TS01 and TS02 are delivered to the sample and can interact with the entire sample. Probes TS01 and TS02 specifically bind to their respective targets if the targets are present in the sample. Unbound probes are removed, e.g., by washing. The address tag 510 is delivered to the biological sample according to a spatial distribution as shown at 530. For example, the address tag is bound to a probe in the sample that specifically binds to biological target 1 or biological target 2 by ligation (or by extension after ligation). The coupled structures are eluted (or "probe/address tag conjugates") from the biological sample and combined. In certain embodiments, if a sequencing adaptor is not included in the address tag or probe/address tag conjugate, a sequencing adaptor may be added, for example by PCR or ligation. The probe/address tag conjugates are sequenced, for example, by high throughput or next generation sequencing methods.

The set used to generate the test products is shown at 540. For example, the presence of product "a 1T2b 1" in the pool indicates that a reading for TS02 was taken at position (a1, b1), and thus target 2 was detected at position (a1, b 1). Thus, sequence readings for TS01 are obtained only at positions (a4, b1), (a4, b2), (a1, b3), (a2, b3), (a3, b3), (a4, b3), and (a4, b4) (positions indicated by horizontal lines of spatial distribution 550), and sequence readings for TS02 are obtained only at positions (a1, b1) (positions indicated by vertical lines of spatial distribution 550). Sequence reads for TS01 and TS02 were obtained at positions (a2, b1), (a3, b1), (a1, b2), (a2, b2) and (a3, b2) (positions shown by the cross-hatching of spatial distribution 550). No sequence reads were obtained for TS01 or TS02 at positions (a1, b4), (a2, b4) or (a3, b4) (unshaded positions of spatial distribution 550). Thus, in the biological sample, target 1 is detected at the majority and bottom of the left side of the sample, whereas target 2 is detected only at the top left portion of the sample, and no target is detected at the top right portion of the biological sample. Differential expression of two biological targets can be mapped back into the biological sample and into the cell types or biological structures at these locations in the biological sample.

In addition to obtaining positional information, the abundance of biological targets at multiple sites in a sample can also be obtained. For example, if the number of a4T1b1 sequences present in the data set is 10 times the number of a4T1b2 sequences, then this indicates that target 1 is 10 times more abundant at position (a4, b1) than at position (a4, b 2).

In the case of nucleotide analysis as shown in FIG. 3, only 2n probes are required for n targets by directly linking the address tags to the probes.for example, analysis of 100 different targets at 10,000 sites in a sample requires 2 × 100 probes and 2 × 100 address tags bound to the probes.the total number of oligonucleotides analyzed is only 400 (200 probes and 200 address tags), the universal primers are not counted.conversely, if the address tags are not decoupled from the probes, the number of oligonucleotides analyzed will be (n × X positions) + (n × Y positions) or, in the above example, 20,000 oligonucleotides, the universal primers are not counted.

The disclosed method and analysis system are particularly well suited for generating large amounts of information with a limited number of analyses. For example, analysis of 5 or more biological targets at 5 or more locations in a sample results in 25 or more compositions. Using digital sequencing reads, the optimal number of sequences to read per combination depends on the sensitivity and dynamic range required and can be adjusted. For example, for each combination averaging 100 readings, the total number of 25 combinations is 2500 readings. If 1,000 targets at 1,000 sites are analyzed with an average sampling depth of 1,000, then 109 reads are required. These numbers, although large, are within the capabilities of the inherent parallel digital sequencing methods, which can produce billions or even trillions of reads in a reasonable amount of time and at a reasonable cost. Thus, by altering the number of sites or the number of biological targets being analyzed, or both, and applying digital sequencing techniques, a vast amount of information can be obtained. In particular aspects, multiple site analysis is performed on two or more biomolecules.

Thus, the present invention provides the ability to simultaneously view many different biological targets at many sites in a sample, e.g., in the same reaction. In some embodiments, the products of the plurality of biological targets and the plurality of sites in the biological sample being analyzed are greater than about 20. In other embodiments, the plurality of biological target products and the plurality of sites in the sample that are analyzed are greater than about 50. In other embodiments, the products of the plurality of biological targets and the plurality of sites in the sample being analyzed are greater than about 100, greater than about 500, greater than about 1,000, greater than about 10,000, greater than about 25,000, greater than about 100,000, greater than about 500,000, or greater than about 1,000,000. It will be appreciated that even greater numbers are contemplated. For example, analyzing 10,000 targets per 10,000 sites in a sample will yield 108 different analyses. In some embodiments, a sufficient number of sites in a sample can be analyzed to achieve a level of resolution at which individual cells are analyzed. Moreover, in embodiments using high throughput digital sequencing, the sequences of at least 1,000 probes or probe/address tag conjugates are typically detected in parallel. More typically, using numerical reads, it is desirable to obtain multiple sequence reads (defined by the target and site, e.g., by the target recognition tag and address tag) for each analysis. An average of at least 3 replicates per assay is obtained, more typically at least 10 and replicates or at least 30 replicates per assay, depending on the design of the experiment and the requirements of the assay. For quantitative readings within a suitable range, it is desirable to take at least 1,000 readings per assay. Thus, if 1,000,000 analyses are performed, the number of sequence reads may be 10 billion or more. Using high throughput sequencing techniques, allowing for redundancy, at least 10,000 probe or probe/address tag conjugate sequences can be detected in parallel, or at least 100,000, 500,000, 1,000,000, 10,000,000,100,000,000, 1,000,000,000 or more probe or probe/address tag conjugates sequences can be detected in parallel.

In certain aspects, disclosed herein are methods and assay systems for estimating differences in the quantity and/or activity of a biological target between samples and/or different sites between samples. In one embodiment, the method comprises estimating the difference in the number of biological targets at each site in the biological sample. In another embodiment, the method comprises comparing the spatial distribution of the abundance and/or activity of one or more biological targets of the plurality of samples.

In-situ analysis of spatial encoding

In certain embodiments, it is desirable to correlate the spatial distribution of target polynucleotide expression, e.g., mRNA expression patterns in a 2D sample having histological features of the sample. In some aspects, the histological features can include expression patterns of known markers of the sample, e.g., tissue specific markers, cell type markers, cell line markers, cell morphology markers, cell cycle markers, cell death markers, developmental stage markers, stem cell or tissue cell markers, differentiation state markers, epigenetic markers, physiological or pathophysiological markers, state change markers, cancer markers, or any combination thereof. In certain aspects, the histological features comprise tissue morphology, e.g., as indicated by an expression pattern of a protein marker. In certain embodiments, imaging techniques may be used in order to obtain information about the distribution of the sample, e.g., histological characteristics of the sample, expression distribution of protein markers, and/or tissue morphology. For example, immunohistochemical Imaging (IHC) and/or immunofluorescence Imaging (IF) may be required.

In certain aspects, the methods provided herein are referred to as spatially encoded protein in situ analysis (SEPIA) for multiplexed in situ analysis of proteins. In some embodiments, SEPIA and related analysis systems can acquire spatial information of the relative abundance of many proteins on a tissue section. In certain embodiments, the disclosed methods and assay systems are based on the use of antibodies (or other affinity capture agents capable of specifically binding to a target, rather than by complementarity of nucleotide sequences) that are labeled with recognition tags that recognize the target protein or antibody, and that can recognize one or more address tags at each of a plurality of sites in a sample. In one embodiment, at least two address tags are provided per site, one of which identifies a location on a tissue section in one dimension (e.g., X-coordinate) and the other of which identifies a location on a tissue section in another dimension (e.g., Y-coordinate).

In any of the embodiments disclosed herein, the biological target may be a peptide or protein, and the methods or assay systems of the invention may be used to assay for the presence of antibodies, activity of enzymes and other proteins, post-transcriptional modifications, active and inactive forms of peptides, and isoforms of peptides in a sample. Thus, the probe may comprise an active region of an enzyme, a binding domain of an immunoglobulin, a defined domain of a protein, a complete protein, a synthetic polypeptide, or the like, such as a mutated peptide, a gamete, or the like.

In any of the embodiments disclosed herein, the probe can comprise a substrate for an enzyme or zymogen, e.g., a kinase, a phosphatase, a zymogen, a protease, or a fragment thereof. In certain aspects, the probes may comprise a phosphorylated substrate for detecting a protein involved in one or more signal transduction pathways. Alternatively, the probe may comprise a specific protease substrate, which is associated with an individual protease or a specific type of protease. In other aspects, the probe may comprise regions of different processing formats, isoforms, and/or enzymes. In certain embodiments, the protein-based probes may be conjugated or linked to oligonucleotide address tags. In a preferred embodiment, the oligonucleotide address tag may comprise a nucleotide sequence component that allows recognition of the protein probe.

In preferred embodiments, the antibodies that bind to the oligonucleotides are compatible with the address tagging scheme disclosed herein. In certain aspects, the methods and assay systems of the invention are highly multiplexed, scalable and high throughput, the methods being useful for detecting the abundance, and/or spatial distribution of activity, of a target protein at multiple sites in a sample, employing nucleic acid reads and independent imaging of the target protein. In preferred embodiments, the methods and assay systems provided herein combine nucleic acid expression patterns (e.g., DAN or RNA expression profiles) with cell-specific type protein marker abundance without the need for protein marker imaging, e.g., immunohistochemical imaging or immunofluorescence imaging. In a preferred embodiment, the spatial resolution of the methods and analysis systems of the present invention approximates the size of an individual cell. In certain aspects, 2D and 3D profiles of RNA associated with proteinometric abundance are generated using the methods and analysis systems of the invention.

As shown in fig. 6, in one aspect, a highly multiplexed protein detection assay is performed (as shown in fig. 6C) on sample 616. In a preferred embodiment, the sample 616 may be a paraffin-embedded or a frozen fresh tissue section that is fixed to a slide. Fig. 6A shows that two probes 620 and 622, respectively, specifically bind to the protein target of interest 602. The first probe 620 may comprise a target binding moiety 608 associated with the oligonucleotide tag 604. The target binding moiety 608 and the oligonucleotide tag 604 may be coupled or covalently linked. The target binding moiety 608 may comprise any affinity capture agent, such as an antibody that specifically binds to the protein target 602. The probe 620 may further include address tags 624 and 626. For example, using sequencing techniques, tag 626 may be a universal primer site for analyzing product amplification and/or the adaptor that recognizes address tag 624 and/or other regions of oligonucleotide tag 604 and/or probe 620. In some embodiments, tag 626 is coupled or connected or associated with address tag 624, e.g., by way of a connection, an extension, a post-extension connection, or any combination thereof. In one aspect, the associated tag 626 and address tag 624, coupled or linked or associated therewith, are coupled or linked or associated in their entirety with the oligonucleotide tag 604. In further embodiments, for example, on the target-binding moiety 608 and/or the oligonucleotide tag 604, the tag 626 and the address tag 624 may be coupled or linked or otherwise associated with the probe 620, respectively.

As shown in fig. 6, in one aspect, a highly multiplexed protein detection assay is performed (as shown in fig. 6C) on sample 616. In a preferred embodiment, the sample 616 may be a paraffin-embedded or a frozen fresh tissue section that is fixed to a slide. Fig. 6A shows that two probes 620 and 622, respectively, specifically bind to the protein target of interest 602. The first probe 620 may comprise a target binding moiety 608 associated with the oligonucleotide tag 604. The target binding moiety 608 and the oligonucleotide tag 604 may be coupled or covalently linked. The target binding moiety 608 may comprise any affinity capture agent, such as an antibody that specifically binds to the protein target 602. The probe 620 may further include address tags 624 and 626. For example, using sequencing techniques, tag 626 may be a universal primer site for analyzing product amplification and/or the adaptor that recognizes address tag 624 and/or other regions of oligonucleotide tag 604 and/or probe 620. In some embodiments, tag 626 is coupled or connected or associated with address tag 624, e.g., by way of a connection, an extension, a post-extension connection, or any combination thereof. In one aspect, the associated tag 626 and address tag 624, coupled or linked or associated therewith, are coupled or linked or associated in their entirety with the oligonucleotide tag 604. In further embodiments, for example, on the target-binding moiety 608 and/or the oligonucleotide tag 604, the tag 626 and the address tag 624 may be coupled or linked or otherwise associated with the probe 620, respectively.

As shown in fig. 6, in one aspect, a highly multiplexed protein detection assay is performed (as shown in fig. 6C) on sample 616. In a preferred embodiment, the sample 616 may be a paraffin-embedded or a frozen fresh tissue section that is fixed to a slide. Fig. 6A shows that two probes 620 and 622, respectively, specifically bind to the protein target of interest 602. The first probe 620 may comprise a target binding moiety 608 associated with the oligonucleotide tag 604. The target binding moiety 608 and the oligonucleotide tag 604 may be coupled or covalently linked. The target binding moiety 608 may comprise any affinity capture agent, such as an antibody that specifically binds to the protein target 602. The probe 620 may further include address tags 624 and 626. For example, using sequencing techniques, tag 626 may be a universal primer site for analyzing product amplification and/or the adaptor that recognizes address tag 624 and/or other regions of oligonucleotide tag 604 and/or probe 620. In some embodiments, tag 626 is coupled or connected or associated with address tag 624, e.g., by way of a connection, an extension, a post-extension connection, or any combination thereof. In one aspect, the associated tag 626 and address tag 624, coupled or linked or associated therewith, are coupled or linked or associated in their entirety with the oligonucleotide tag 604. In further embodiments, for example, on the target-binding moiety 608 and/or oligonucleotide tag 604, the tag 626 and address tag 624 may be coupled or linked or otherwise associated with the probe 620, respectively

In certain embodiments, primary and secondary antibodies may be employed. For example, the target-binding moiety 606 and/or the target-binding moiety 608, do not specifically bind directly to the target 602, but may specifically bind to a primary antibody that specifically recognizes the target 602. In this case, the target-binding moiety 606 and/or the target-binding moiety 608 may be comprised in a secondary antibody. In certain aspects, methods involving a primary antibody and a secondary antibody are suitable, when target expression in the sample is low, because one molecule of the target 602 may bind to multiple molecules of the primary antibody, thereby enhancing the signal.

In other embodiments, as shown in fig. 6D, the biological target 632 is analyzed according to a combined address tagging scheme. The two probes 650 and 652 specifically bind to the biological target of interest 632. In one embodiment, the first probe 650 comprises a target binding moiety 638 associated with the oligonucleotide tag 634 and the second probe 652 comprises a target binding moiety 636 associated with the oligonucleotide tag 640. The target-binding moiety 638 and the oligonucleotide tag 634 (or the target-binding moiety 636 and the oligonucleotide tag 640) may be bound or covalently linked. In a particular embodiment, the target-binding moiety 638 or 636 comprises an affinity capture agent, e.g., an antibody that specifically binds the target 632. In certain embodiments, target 632 comprises a protein moiety, an oligosaccharide or polysaccharide moiety, a fatty acid moiety, and/or a nucleic acid moiety. In some embodiments, each probe has a portion that specifically binds to the linker 662, e.g., by specific nucleic acid hybridization. In one embodiment, oligonucleotide tag 634 or 640 (or a portion of oligonucleotide 634 or 640) specifically hybridizes to the linker. The linker is capable of specifically binding to the following compositions:

1) a 5 'portion of oligonucleotide tag 634 and a 3' portion of oligonucleotide tag 640;

2) a3 'portion of oligonucleotide tag 634 and a 5' portion of oligonucleotide tag 640;

3) a 5 'portion of oligonucleotide tag 634 and a 5' portion of oligonucleotide tag 640; or

4) A3 'portion of oligonucleotide tag 634 and a 3' portion of oligonucleotide tag 640. In certain embodiments, the oligonucleotide tag 634 or 640 is a linear molecule, a branched molecule, a circular molecule, or a combination thereof. After both probes bind to the biological target and the linker binds to both probes, an address tag can be delivered to the sample and coupled to the linker. For example, the adaptor may be labeled with address tag 654 associated with tag 656 and/or labeled with address tag 658 associated with tag 660. Tags 656 and 660 can be universal primer sites or sequences that can recognize address tags and/or other regions of the adapter/address tag conjugates used to amplify the assay products, for example, by employing sequencing techniques. In certain embodiments, the address tags are labeled at the same or different ends of the linker. In other embodiments, the address tag and/or tags 656 or 660 can be bound to the linker before the linker/address tag or linker/tag conjugate or complex is delivered to the sample to bind to the probe bound to the biological target.

In certain embodiments, for example, all or part of the linker/address tag conjugate sequence is detected by nucleic acid sequencing. In other embodiments, all or part of the oligonucleotide tag sequence, and/or all or part of the linker/address tag conjugate sequence, is detected. For example, a first address tag may be coupled to the oligonucleotide tag 634 and a second address tag may be coupled to the linker 662. The duplex formed between oligonucleotide tag 634 and the linker 662 can be used for extension and sequencing to generate a conjugate comprising the sequence of the first address tag, all or part of oligonucleotide tag 634, all or part of the linker 662 and the second address tag.

The labeling scheme is not limited to the use of two or more probes for the same biological target. For example, where the probe is labeled, a tag (e.g., an address tag, the linker used for ligation, or a universal sequencing primer or amplification primer sequence) can be coupled to the linker of the specific binding probe, rather than to the probe itself.

Further details of the polynucleotide-protein conjugates of the present invention are also disclosed in U.S. provisional patent application No. 61/902,105 filed on 2013, 11/8/2013 entitled "polynucleotide conjugates for analyte detection and methods therefor," the contents of which are hereby incorporated by reference in their entirety.

In some embodiments, more than one of the linkers is used. For example, a pair of such linkers are used to specifically bind oligonucleotide tags 634 and 640, respectively. In certain embodiments, the pair of linkers are DNA molecules: 1) hybridized to or bound to a protein-DNA conjugate, e.g., probes 650 or 652; 2) having free 3 'and/or 5' ends such that coding sequences (e.g., address tags 654 and 658) are added in subsequent steps, e.g., by ligation; 3) in a connectable form if they are co-located or in close proximity to each other. In some embodiments, a portion of the oligonucleotide portion of probe 650 or 652 acts as a splint to enable ligation or extension and ligation of the pair of linkers. Additional tags (e.g., address tags, the adaptors used for ligation, or universal sequencing primers or amplification primer sequences) are bound to the adaptors generated by binding the adaptor pairs.

In other embodiments, the disclosed methods allow for simpler protein-based manipulation than nucleic acid-based analysis. For example, the linker can be designed so as to be compatible with the same encoding oligonucleotide used in a nucleic acid-based assay, e.g., an RNA-based assay. Thus, both types of binding assays (detection of protein targets with protein-polynucleotide conjugates and nucleic acids with nucleic acid probes) can be performed in the same reaction or in the same experimental procedure, and spatial addresses can be performed on both probes simultaneously.

In yet another embodiment, the invention provides controls for the analysis of a biological target that detects a protein target or comprises a protein moiety, e.g., the nucleic acid moiety of a protein-nucleic acid conjugate is used to hybridize with nucleic acids in a sample, this anchors an "artificial" protein in a sample (known composition and abundance based on the abundance of hybridized sequences) that can be detected by several methods, including the methods disclosed herein for the analysis of protein-binding specific markers, the methods are not limited to proteins, e.g., small molecules, such as haptens, can also be used in one aspect, fig. 6E illustrates the general concept of a method for detecting RNA using known compositions and abundances in a sample, thereby providing controls for the detection of other targets (e.g., proteins) in a sample fig. 6E, conjugates 662 and 664 each comprise a nucleic acid moiety and an antibody-binding moiety (circular for the antibody-binding moiety of conjugate 662, triangular for the antibody-binding moiety of conjugate 664) in a sample, RNA having known compositions and abundances is detected by the nucleic acid moiety of the antibody of the conjugate 662 in the present examples, the invention, antibodies are specifically binding to HA-RNA-binding proteins found in the present examples, antibodies are found to HA-AG-binding proteins, or antibodies found in the present invention, antibodies are found in the known antibodies, AG-binding domains, or antibodies-AG-binding proteins.

FIG. 6B shows an address tag scheme that may be used for 100 individual sites in a sample. For example, 20 probe/address tag conjugates X1-X10, Y1-Y10 were used, each of X1-X10 comprising address tag 624, and each of Y1-Y10 comprising address tag 628. The address tags contained in each of X1-X10 and each of Y1-Y10 may be uniquely identified. For example, probe/address tag conjugate X9 is delivered to the biological sample in the ninth vertical channel in 612. However, the "X" probe/address tag conjugate is delivered to the biological sample in the tenth vertical channel and the "Y" probe/address tag conjugate is delivered to the biological sample in the tenth horizontal channel, as shown at 614. For example, probe/address tag conjugate Y1 is delivered to the biological sample in a first horizontal channel at 614. In other embodiments, the "X" tag may refer to the "a" tag and the "Y" tag may refer to the "b" tag.

Fig. 6C shows a representative tissue slice 616 consistent with grid 618. Arrows indicate how the "X" probe/address tag conjugates and the "Y" probe/address tag conjugates are delivered on grid 618 in line with tissue section 616. For example, if the probe/address tag conjugates associated with the targets, X9 and Y1, once analyzed, then the targets are present in the tissue section at positions (X9, Y1).

The structure of any suitable oligonucleotide/antibody (or other target-specific binder) conjugate can be used to convert specific protein detection into nucleic acid analysis. In certain embodiments, for example, as shown in fig. 7A, probe 708 specifically binds to protein target 702. The probe 708 may comprise a target binding moiety 704 associated with an oligonucleotide. The target binding moiety 704 and the oligonucleotide tag 706 may be bound or covalently linked. The target binding moiety 704 may comprise any affinity capture agent, such as an antibody that specifically binds to the protein target 702. Probe 708 may further comprise an "X" address tag 710 and a "Y" address tag 712. The address tags 710 and 712 may be bound to universal primer sites for amplification of assay products and/or adaptors (not shown in FIG. 7) to identify the address tags 710 and 712 and/or the oligonucleotide tags 706 and/or other regions of the probes 708, for example, by sequencing techniques. The combination of different tags may be accomplished by linking, extending, post-extending, or any combination thereof. In some embodiments, the address tags 710 and 712 are bound to one side or the other side (e.g., the 5 'or 3' end of the sequence) of the oligonucleotide tag 706, respectively. In further embodiments, the address tags 710 and 712 may be combined with 5 'or 3' of the oligonucleotide tags 706. For example, the address tags 710 and 712 may be directly or indirectly bound, and the address tags 710 and 712 may be directly or indirectly bound to the 5 'or 3' of the oligonucleotide tag 706.

In other embodiments, for example, as shown in fig. 7B, the probe 720 specifically binds to the protein target 714. The probe 720 may comprise a target binding moiety 716, binding, ligating or otherwise associating an oligonucleotide tag 718. The target-binding moiety 716 can comprise any affinity capture agent, such as an antibody that specifically binds to the protein target 714. The probe 720 may further comprise an oligonucleotide sequence 722 that specifically hybridizes to the oligonucleotide tag 718. In one embodiment, sequence 722 is complementary to oligonucleotide tag 718. The sequence 722 may incorporate an "X" address tag 724 and a "Y" address tag 726. For example, by sequencing techniques, the address tags 724 and 726 may bind to universal primer sites and/or the adaptors (not shown in fig. 7) used to amplify the assay products to identify the address tags 724 and 726 and/or the sequence 722 and/or other regions of the probe 720. The combination of different tags may be accomplished by linking, extending, post-extending, or any combination thereof. Like FIG. 7A, address tags 724 and 726 are bound, directly or indirectly, to either side of oligonucleotide sequence 722 (e.g., the 5 'or 3' end of the sequence), respectively.

In a further embodiment, for example, as shown in FIG. 7C, a form of "2-antibody" may be used. For example, in fig. 6, the "2-antibody" format is similar to the dual target approach mentioned above. In this embodiment, two antibodies that specifically bind to the protein target are bound to one oligonucleotide, either directly or indirectly to the "X" address tag and the "Y" address tag and the universal primer site for amplification of the assay product and/or the adapter for sequencing. In some embodiments, the two antibodies may bind different epitopes or sites on the protein target. In preferred embodiments, both antibodies are required to bind to the protein target to generate a signal, providing greater specificity than using only one antibody. It is also contemplated that more than two antibodies may be used in conjunction with the oligonucleotides and the methods and assay systems of the invention may be used.

As disclosed herein, the methods and analysis systems of the present invention allow for a higher level of multiple analyses. In one embodiment, the probe may be delivered to the entire surface of the 2D sample in a batch process and then the address is labeled by delivering an address label in a spatially defined pattern. For example, two sets of address tags ("X" address tag and "Y" address tag) are employed in a combination as discussed above. Once the analysis is complete, the analysis product is eluted and sequenced. The address tag sequencing information identifies the location where the analysis is performed and the probe sequence information (identifying the target protein tag) identifies the protein that is the target. In one aspect, digitized readings of the frequency of a particular assay product (e.g., sequencing product) can be used to infer the relative abundance of a target in a sample. This information may be correlated with other information, including conventional histological information, and/or transcript abundance obtained by relevant Spatially Encoded Gene Analysis (SEGA). In preferred embodiments, the methods and assay systems of the present invention do not rely on imaging techniques for obtaining spatial information about the target protein. Rather, in preferred embodiments, the spatial distribution of the abundance and/or activity of the target protein may be determined by sequencing.

In one embodiment, for integrin gene expression analysis, the address tag scheme is compatible with and can be used in both types of analysis. For example, the same composition of "X" address tags and "Y" address tags can be labeled onto antibody-DNA conjugates of the target protein, and can be labeled onto probes of the target polynucleotide sequence, at each of a plurality of sites in the sample. In one embodiment, the target polynucleotide or its complementary nucleotides encodes all or part of the target protein. Thus, for each site in the sample, the abundance and/or activity of the target protein and its corresponding polynucleotide can be detected by analysis of the sequencing product using the same address tag. In a preferred embodiment, the step of analyzing the binding of the probe or probe/address tag conjugate to the target protein and the step of analyzing the binding of the probe or probe/address tag conjugate to the target polynucleotide may be performed simultaneously in the same reaction. In other embodiments, different address tags may be coupled to antibody DNA conjugates of the target protein and to probes of the target polynucleotide to detect the abundance and/or activity of the target protein and target polynucleotide at a given site. The results of the analysis of the target protein and target polynucleotide for each site in the sample are then integrated.

A variety of methods can be used to form the amplification structures, for example, ligation by proximity probes followed by sequential ligation by a pair of spatially encoded adaptors (address tags). In one embodiment, two DNA probes hybridize adjacent to each other on one RNA target (or template). The probes are then ligated to each other and the number of ligated pairs is used as a measure of the number of targets present in the sample. In some cases, however, the efficiency of T4DNA ligase may be reduced when ligation occurs on an RNA template as compared to on a DNA template. In other cases, the efficiency of the ligation reaction depends on the sequence of the DNA probe being ligated, and in particular on the first few bases on either side of the junction. In some embodiments, the methods of the present invention alleviate both of these problems. In this case, rather than using probes that hybridize in direct proximity on the RNA target, the probes are separated by a distance by having non-hybridizing overhang sequences adjacent to them. These overhang sequences are designed to be complementary to a short DNA splint. The splint is generic to all probe pairs of the multiplex assay or specific to a given probe set or subset of probe sets. The distance of the two probes in a set can be adjusted to optimize the efficiency of the ligation. This degree of flexibility in distance provides additional degrees of freedom in the design of the probe relative to the use of proximity probes. Once the probe has hybridized to the RNA target, excess probe is washed away. The splint hybridizes to the overhang region adjacent to the probe, and the probe is added by ligase. The remaining experimental steps are performed after ligation, e.g., attaching a space-coding linker to each end of the ligated probe pair. In certain aspects, the methods of the invention increase the efficiency of ligation of two such probes, since ligation of DNA splints is more efficient than ligation of RNA splints. Furthermore, the use of a universal splint eliminates variations in sequence-dependent ligation efficiency between multiple probes in a multiplexed in situ assay. On the other hand, since the degree of freedom in the change of the distance between the probes is improved, the probes can be designed more easily, and a more suitable probe set can be designed.

Reagent delivery system

The disclosed reagent delivery system includes an instrument that allows reagents to be delivered to discrete portions of a biological sample, maintaining the integrity of the spatial arrangement in the addressing scheme. The reagent delivery system of the assay system of the present invention includes optional imaging devices, reagent delivery hardware and control software. Reagent delivery can be accomplished in a variety of ways. It should be noted that the reagents may be delivered to a plurality of different biological samples simultaneously. Although a single tissue section is illustrated here, multiple biological samples can be manipulated and analyzed simultaneously. For example, a series of sections of a tissue sample may be analyzed in parallel, and the data combined to construct a 3D map.

The techniques for formulating and delivering biomolecules (e.g., oligonucleotides or antibodies) and chemical reagents (e.g., small molecules or dNTPs) are well known in the art, and the use of such an instrument system is well known to those skilled in the art and readily applicable to the detection system disclosed herein, one example of which is a suitable reagent delivery system is L abcyte^TMAn Echo acoustic liquid processor that can be used to deliver nanoliter-scale droplets of highly accurate and reproducible biomolecules. One skilled in the art can incorporate such reagent delivery devices into an overall system in which software is used to specify the location to which the reagent is delivered.

Other instruments that can be used to deliver reagents and/or encode identifiers on biological samples include, but are not limited to, ink jet printing, mechanical printing using a needle, pen, or capillary, micro-contact printing, photochemical or photolithographic methods, and the like. In some applications, it may be preferable to segment or isolate certain regions of a biological sample into one or more analysis regions for different reagent distributions and/or biological target detection. The analysis areas may be physically separated using obstacles or channels.

In an exemplary aspect, the reagent delivery system may be a flow-based system. In the present invention, a flow-based system for reagent delivery may include instruments such as one or more pumps, valves, reservoirs, channels, and/or reagent storage units. The reagent delivery system is configured to move the fluid to contact discrete portions of the biological sample. The movement of the reagent may be driven by a pump, e.g. downstream of the fluid reagent. The pump may drive the flow of each fluid reagent to (and through) the reaction zone. Alternatively, the reagent may be driven by the gravity of the fluid. US pub. nos.20070166725 and 20050239192 disclose some general purpose applied fluidics tools that can be used in the analytical system of the present invention, which allow precise manipulation of gases, liquids and solids to accomplish very responsible analytical operations with relatively simple hardware.

In a more specific example, one or more fluidic units may be attached to the above-described basis-bias immobilized biological sample. The fluidic unit comprises connected inlet and outlet tubes and an optional external pump for delivering reagents to the fluidic unit and through the biological sample. The fluidic unit is configured to deliver reagents to only certain portions of the biological sample, limiting the amount and type of reagents delivered to any particular portion of the biological sample.

In other aspects, the microfluidic device may be integrated onto a substrate, on which the biological sample is processed or attached on top of the substrate. Microfluidic channels for storing and transporting fluids may be formed on and/or over a planar substrate by a layer of fluid proximate to the substrate. The fluid reagent may be selected and delivered according to the selective opening and closing of valves between the reagent containers.

Pumps typically include structure to move the liquid and/or the reagents in the liquid. In some examples, the pump may be configured to move liquids and/or reagents through small volume channels (e.g., microfluidic structures). The pump may be operated mechanically by applying positive or negative pressure to the liquid and/or the structure carrying the liquid, electrically by applying an electric field, or both mechanically and electrically. Typical mechanical pumps may include syringe pumps, peristaltic pumps, rotary pumps, compressed gas, pipettes, and the like. For example, external syringes and pneumatic pumps may be used to inject the liquid and create a flow of the liquid in the microfluidic device. The mechanical pump may be a miniature machine, a model, or the like. A typical electrokinetic pump may include electrodes and may operate by electrophoresis, electro-endosmosis, electrocapillary, dielectrophoresis (including the form of traveling waves it produces), and/or the like.

Valves generally include structure for regulating the passage of fluid therethrough. The valve may include, for example, a deformable structure that can be selectively deformed to partially or completely close a channel, a movable protrusion that can be selectively extended into the channel to partially or completely block a channel, an electrocapillary structure, and/or the like.

An open gasket may be attached to the top of the biological sample and the sample and reagents may be injected into the gasket. Suitable gasket materials include, but are not limited to, neoprene, nitrile, and silicone rubber. Alternatively, a water-tight reaction chamber may be formed by a gasket sandwiched between a biological sample on a substrate and a chemically inert, water-resistant material such as, but not limited to, black anodized aluminum, a thermoplastic (e.g., polystyrene, polycarbonate, etc.), glass, etc.

Microfluidic devices that can be used in the disclosed methods and systems are disclosed in detail in application serial No. 61/839,320, filed 2013 on 25/6 entitled "spatially encoded biological assays using microfluidic devices," in international application No. PCT/US2014/___, filed 2014 on 25/6, attorney docket No. 699932000340, entitled "spatially encoded biological assays using microfluidic devices," which is incorporated herein by reference in its entirety for all purposes.

In an alternative embodiment, the analysis system comprises an imaging modality that detects characteristics and tissues of the biological sample of interest. The images obtained, for example, can be used to design the delivery pattern of the agent. The imaging modality is optional and may be used as an individual imaging modality in lieu of, for example, observing a biological sample with a microscope, analyzing tissue of a biological sample, and assigning a spatial distribution for delivery of an analytical reagent. If included, the delivery system may comprise a microcircuit device including an imager, such as a CCD or IGFET-based (e.g., CMOS-based) imager and an ultrasonic nebulizer for reagent delivery as described in US Pub.No.20090197326, which is incorporated herein by reference. It should be noted that although an X-Y grid configuration is illustrated herein, other configurations may be used, for example, according to the layout of the tissue sample, according to the cell-specific groupings, cell layers and/or cell types in the tissue, and the like.

In yet another alternative, semiconductor technologies such as masking and spraying techniques may be used in conjunction with the reagent delivery methods and systems disclosed herein to control the delivery of reagents to specific locations on the surface of a biological sample. Specific areas of the biological sample may be protected from exposure to the reagent by using masking techniques. The reagents are introduced into the biological sample by conventional techniques such as spraying or liquid flow. The use of masked transfer creates a patterned transfer scheme on the substrate surface.

In one aspect, the reagent delivery apparatus is based on inkjet printing technology. There are a variety of different ink jet mechanisms (e.g., thermal, piezoelectric) and exhibit compatibility with aqueous and organic ink formulations. Independently driven nozzle sets can be used to deliver multiple reagents simultaneously and achieve high resolution.

To locate specific sites of interest, the informative image of the analyzed biological sample can be used to assist in the reagent delivery method and associated coding scheme. The sample region of the biological sample may be identified by image processing integrated with other features of the analysis system (e.g., images of cell types differentiated by immunohistochemistry or other staining chemistry). In some aspects, the software automatically converts the image information into a reagent delivery pattern. In certain embodiments, the mechanism by which biological samples are very accurately recorded and arranged for reagent delivery is an important component of the assay system. Such as the application of slide fiducial markers and/or other very accurate physical positioning systems, are employed for this purpose.

The method and analysis system of the present invention may comprise a complete software suite customized for the method and analysis system. Alternatively, oligonucleotide design software is used to design the encoding nucleotides (target-specific oligonucleotides in the embodiment where nucleic acids are analyzed) for the particular assay to be performed, and may be integrated as part of the system. Alternatively, algorithms and software for reagent delivery and analytical data (e.g., sequence analysis) may be integrated for detecting the analytical results. Integrated data analysis is particularly useful, and depending on the scale, the type of data set generated may be extensive. Algorithms and software tools specially designed for analysis of relevant spatial data generated by the analysis system, including pattern analysis software and visualization tools, improve the value of the data generated by the analysis system.

In certain aspects, the assay system comprises methods to establish and implement reagent quality control, such as sequence integrity and fidelity in pools of oligonucleotides. In particular, reagents are formulated based on factors such as volatility, stability at critical temperatures, and chemical compatibility with compatible reagent delivery instruments, and are analyzed by instruments integrated in the analysis system.

Applications of detection systems

It will be apparent to those skilled in the art, upon reading this disclosure, that there is a vast array of biological studies, diagnostics, and important areas of drug development that benefit from high throughput multiplex analysis systems that can simultaneously measure the number and location of biological targets in a biological sample. For example, the ability to estimate the relative abundance of different RNA transcripts, combined with the ability to reconstruct a spatial distribution of abundance at many locations in a tissue, which may be a single cell or even smaller, may enable different areas of basic research. The following are exemplary applications and are not meant to limit the scope of protection. .

In one embodiment, the disclosed analysis system and apparatus can discriminate between different tissue types based on tissue-specific differences in gene expression. In one aspect, the disclosed assay systems and devices can be used to analyze and discriminate between mRNA and hnRNA, and thus can be used for parallel analysis of RNA processing in situ. In one aspect, the probes are designed to target introns and/or exons. In one aspect, the internal probe signals from hnRNA, rather than mRNA. The gDNA background signal can be detected by selective pretreatment with DNase and/or RNase. In some aspects, splice site specific probes selected for spliced RNAs are designed and used. In certain embodiments, a combination of internal probes, external probes, and/or splice site specific probes may be used to identify the relative levels of processing intermediates and differences between different cells in a tissue section. In general, RNA can bind to different types of proteins, and in particular, hnRNA is complexed with proteins to form hnRNP (heterogeneous ribonucleoprotein). In one embodiment, the apparatus and analysis system of the present invention can be used for highly parallel in situ experimental footprinting experiments. In some aspects, probes can be closely arranged with a smaller amount of RNA to produce a signal profile along the molecule than if 1000 different RNAs were targeted. Relative changes between cell types in this profile may indicate differences in the availability of the RNA at specific sites of analysis.

In one example, 3-dimensional patterns of gene expression are detected by analyzing a series of tissue sections, somewhat analogous to image reconstruction from a CT scan. Such methods can be used to measure changes in gene expression in disease pathology, e.g., cancerous and/or damaged tissue, inflammation, or infection. Using the analysis system of the present invention, more specific information on gene expression and protein localization in complex tissues is obtained, allowing new insights into the function and rules in normal and diseased states, and providing new hypotheses that can be tested. For example, the analysis system of the present invention can integrate new insights at the organizational level obtained from many studies of multiple subjects and studies of larger items such as ENCODE (Birney, et al., Nature, 447: 799-. The analysis system also facilitates computational work in model interaction networks for gene expression in the field of system biology

The assay system also provides novel methods for assaying somatic variations, e.g., for somatic mutations or changes in cancers of infectious organisms. For example, tumors are highly typical isoforms that include cancer cells and genetically normal cells in a locally abnormal environment. Cancer cells undergo mutation and selection, and it is not uncommon for local cloning to progress in this process. The identification of relatively rare somatic mutations in the tumor environment facilitates the role of key mutations in clonal variation selection. The transcriptional patterns associated with angiogenesis, inflammation, or other cancer-related processes can be analyzed in both cancer and genetically normal cells to gain an understanding of cancer biology and aid in the development of new drugs for treating cancer. In another example, individuals have different sensitivities to the infecting organism, and the assay system of the present invention can be used to study interactions between a microorganism and a tissue or between multiple cell types in a tissue.

Importantly, in addition to providing spatially correlated information, the present invention greatly increases the sensitivity of detecting rare mutations, and the emitted signal can be increased more dramatically because only a small number of positions are available for analysis in a given reaction. In a typical assay for rare mutations in a mixed sample, the sample is processed in batches, e.g., nucleic acids are extracted from a number of cells and placed in a single laboratory. Thus, if a mutation is present in one of the 10,000 cells, it must be detected in the background of normal DNA from the 10,000 cells. In contrast, many cells can be analyzed using the analysis system of the present invention, but individual cells or small populations of cells will be identified by the spatial coding system. Thus, in the analysis system of the present invention, the background is reduced by orders of magnitude, which greatly increases the sensitivity. Furthermore, spatial organization of the mutated cells can be observed, which is particularly important for detecting critical mutation sites in tissue sections in cancer. Molecular histological analysis has led to new insights into tumor biology, with potential applications in diagnostics. The techniques of the present invention can greatly enhance the effectiveness of such methods.

The following exemplary embodiments and examples are presented to further describe and illustrate various aspects of the invention, but are not intended to limit the scope of the invention in any way, shape, or form, either explicitly or implicitly.

Example 1 verification of concept and extensibility of addressing scheme

An analytical model system was developed that employed multiple spatial coding abundances of polynucleotide targets in validation work using gene chips. The basic design validates the concept of the analysis and the addressing scheme and establishes a usable analysis before solving the problems associated with the analysis of more complex biological samples.

The gene chip is used as a substitute for tissue section. The target sequences of the gene chip are fully specified so that the composition of the target is known and systematically varied. One skilled in the art will appreciate from the present disclosure that similar assays can be performed on different samples, including tissue sections, and for different targets, including polynucleotide or protein targets, as well as other biological targets.

16-fold × 8 locus analysis using 8-layer gene chips as artificial samples

The 16 by × 8 site analysis was performed using a custom DNA gene chip (Agilent) as an artificial sample since 8-layer gene chips are used with the above commercially available 8 sites sixteen different target sequences were analyzed separately with 128 times the amount of DNA.

Example 2 demonstration of spatial coding Using spotted Gene chips

The scalability of the spatial processing and analysis system was demonstrated by implementing the analysis of 24-fold × 24-sites using a chip model system.

The amount of biological target, here the DNA target sequence, varies systematically at each assay site on the substrate on the gene chip. For example, in a gene chip with a spot size of 50 microns (center-to-center), a 1mm2 area contains-400 spots. The surrounding area of each site is optionally occupied by an area lacking such spots, allowing the target sequence to be resolved separately. Alternatively, 2 or more adjacent spots or spots surrounding a region lacking the target sequence may be clustered.

To confirm that spatial addressing or encoding is accurate, the sites comprising different target compositions show that the assay readout for each site matches the expected composition. Using 24 target sequences, simple digitized patterns of sites with different combinations of 12 targets present and 12 targets not present were made, binary coded (0 absent, 1 present). The detected readings are then determined and displayed, and the detected regions match the spatially decoded expected signal. In this particular embodiment, the coding (address tag) space is large enough (2^24) that a small number of errors do not cause different codes to be confused. Furthermore, this design allows for the identification of errors and allows for the estimation of the accuracy of spatial encoding, but also the accuracy of the estimation of the presence or absence of target sequences.

The ability to detect quantitative differences was assessed by the dose-response curves generated for each of the 24 assays performed at each site in the 24-site assay. This allows the ability to estimate the detection limit, dynamic range and given fold change within the detection range.

In other words, a site has multiple spots, the number of spots assigned to each of the 24 target sequences is variable, and each of the 24 sites may have a different composition.A 1 × 3 inch gene chip is large enough to allow forThis larger set of 24 sequences would require convolution, and this can be done using high throughput techniques, such as the next generation sequencing techniques (e.g., SO L iD)^TMThe use of the 24-fold analysis demonstrated the accuracy of spatial encoding and decoding and the quantitative anti-response of the detection system (L ife technologies, inc., Carlsbad, Calif.), or genomic analysis (Illumina, inc., San Diego, Calif.).

EXAMPLE 3 analytical methods for preserving samples and biological samples

Genomic DNA is tested for the purpose of characterizing coding and regulatory sequence changes, such as Single Nucleotide Polymorphisms (SNPs) or mutations, small insertions and deletions (indels), copy number variations such as gene deletions or amplifications, and gene rearrangements such as transposition, all of which may have significant functional significance in cancer and other diseases. The function of the genomic sequence variation as a position in the sample may be indicative of a somatic chimera in the sample. In cancer samples, mutations can provide prognostic or diagnostic markers that are useful for determining the optimal treatment regimen. Genetic mutations can identify regions of a sample that include cancer cells, which can help to distinguish them from normal cells or cells in a tumor microenvironment, i.e., cells that are normal at the genetic sequence level but otherwise disturbed due to the influence of cancer cells. However, in order to distinguish the signal derived from the DNA target from the signal derived from the RNA target, the probe may be designed to hybridize to a non-coding sequence that is not transcribed. In addition, to ensure the specificity of the DNA target, RNA can be degraded by rnase treatment. Genomic DNA is also analyzed to obtain information on its organization and to provide information on the activation state of specific genes. For example, the ability of a probe to bind to DNA can be used to indicate whether the DNA is concentrated or unbound or whether the DNA is in an open form for transcription. This type of determination may benefit from comparative analysis of samples in which genes are differentially active. Similarly, it may be useful to correlate information about the abundance of RNA and/or protein proteins with information about the activation state of the gene. Other types of information are obtained from epigenetic marker analysis associated with genomic DNA, such as methylation status and the presence and modification of histones and other proteins.

The handling of the absolute number of product molecules produced by very small or compromised samples is enhanced to address the problem of low recovery efficiency; that is, elution is effective, avoiding loss of molecules adsorbed to the surface. The latter problem is solved by including a carrier material, such as glycogen or carrier nucleic acid.

To employ the analysis on biological samples and to make the RNA analysis of tissue sections as informative as possible, pre-stored information is included in the design of the analysis about the transcription of the target in a range of expression levels to abundance in a particular tissue. High abundance transcripts, as well as some moderate and low abundance transcripts, were targeted to facilitate initial assessment of the quantitative performance characteristics of the assay. In this assay, a control RNA template is immobilized on a solid support to create an artificial system. The assay is performed using T4DNA ligase, which can repair nicks in DNA/RNA hybridization. The analysis is performed on matching coverslips or different sections of the same coverslip, in one example genomic DNA is analyzed, and in another example RNA is analyzed. When genomic DNA is analyzed, the cover slip may be pretreated with RNase, and when RNA is analyzed, the cover slip may be pretreated with DNase. The results of the analysis can be determined by extracting genomic DNA or RNA and determining the relative amounts by real-time fluorescent quantitative PCR or reverse transcription real-time fluorescent quantitative PCR.

Example 4 multiplex spatially encoded Polynucleotide abundance analysis

This example describes a representative multiplex spatial coding abundance analysis of polynucleotide targets. One skilled in the art will appreciate from the present disclosure that similar assays can be performed on protein targets as well as other biological targets.

57 Re-analysis of formalin fixed, Paraffin Embedded (FFPE) samples

The approach of using adjacent tag ligation followed by sequential ligation using a pair of spatially encoded adaptors (address tags) is used to form an amplifiable structure. For example, two target-specific probe oligonucleotides are ligated together in situ hybridization. A unique linker or address tag encoding the X position is introduced through the microfluidic channel and attached to the 5' end of the probe. A second address tag encoding the Y position is similarly mounted to the 3' end of the probe. The address tag contains universal primer sites that allow for the installation of additional sequencing adapters by PCR. The final structure serves as a substrate for next generation sequencing.

57 re-analyses were performed on commercial formalin-fixed paraffin-embedded normal human liver and pancreatic tissue (Pantomics) using a 57 target probe pool. The probe pool included probes for 18 liver-specific targets, 19 pancreas-specific targets, 4 housekeeping gene targets, 6 custom-generated negative control sequences, and 10 pluripotency markers. All liver-specific probes were rich in liver, and all but 3 pancreas-specific probes were strongly enriched in pancreas. These 3 probes all have a small total number, so it is likely that they are sequences that hybridize or ligate inefficiently, and therefore are not reported accurately. The results of this experiment are consistent with published data on expression in normal liver and pancreas (BioGPS, available at biology. org/# goto ═ welome).

Many different reagent delivery techniques, including random access methods such as ink jet and pin spotting (pin-spotting), can be used for multiplex assays. Systems using microfluidic flow channel devices have been chosen for several reasons. First, soft lithography typically allows microfluidic devices to be developed quickly and with little expense and time required to develop and purchase suitable instruments for printing or spotting reagents. Second, the size of the sample area can be tightly defined using microfluidic devices, and printed droplets of reagents can be unevenly dispersed on the surface of the FFPE sample, resulting in sample areas of different sizes and shapes. Third, reagent delivery systems using microfluidic devices do not require precise sample calibration. This feature allows for the continuous connection of two encoded position tags (e.g., two address tags). Continuous ligation may reduce the formation of unwanted products compared to simultaneous ligation of two address tags. In some aspects, for reagent delivery techniques using ink jetting and pin spotting, the position of each droplet or site of a first address tag must coincide with the droplet or site of a second address tag in order to form a complete structure in a continuous connection. This requires that an accurate record of the sample is always kept in two printing steps. In contrast, in certain embodiments, methods and analysis systems based on the present invention utilize a pair of microfluidic devices, each having parallel channels, wherein the channels of the first and second devices are oriented perpendicular to each other, as shown in fig. 9A.

The microfluidic addressing device, as shown in fig. 9A, shows a pair of addressing devices in a prominent arrangement, as shown in fig. 9B, a poly (dimethylsiloxane) (PDMS) elastomeric device with 16 × 16 channels and a 100 μm channel width, as shown in fig. 9C, an assembled device with a clip structure and a peristaltic pump structure.

In some aspects, the geometry of the device defines a rectangular array of connection points (or intersections), each connection point having an area defined by the width of two channels. When each first channel and each second channel receives and transmits a different address tag, the result is a unique identification tag pair for each junction or intersection in the array. Fluid flow in microfluidic devices is typically driven by an external syringe pump or vacuum and often requires a complex detection device, including connections between the micro-channels and macro-components of the system. The reagent delivery system used in this example is a self-contained system that loads reagents into the channels. The device can be expelled from the PDMS elastomer and includes a reagent reservoir, and micro-addressing channels, each connected to a larger peristaltic pump channel. The device is applied to the surface of an FFPE sample and held in place. A thumb wheel is applied to the pump channel and a rolling function can draw fluid from each reservoir through the addressing channel where the tissue sample is contacted and the address tag is attached to the hybridization probe. After the first connection, the device is removed and the sample washed. Second, a vertical device is used to mount a second set of address tags. Only the probes that are at the intersection of the two channels can receive both address tags. The device can be cleaned and reused.

A microfluidic device with a 5-site × -site layout nested to match a custom designed tissue gene chip (TMAs) containing a corresponding 5 × checkerboard pattern was produced, the custom designed tissue gene chip containing a corresponding 5 × 5 checkerboard pattern was produced, the custom designed tissue gene chip containing the precision of a commercially available formalin fixed-paraffin embedded normal human liver and pancreas (Pantomics) tissue used in one checkerboard pattern as used in the examples above, known patterns of tissue spots on such an array were used to validate the spatial coding system, fig. 10 shows an immunofluorescence image of a TMA and expression map generated using the detection system of the microfluidic reagent delivery system fig. 10a shows a custom Immunofluorescence (IF) image stained with two liver-specific antibodies PYG L, the specificity of hepatocytes and annexin a2, the specificity of cholangiocytes is a protein map, the bright available reference is liver tissue, the intersection of which is a PYG L, the abundance of hepatocytes and annexin a, the specificity of the annexin a, cholangiocytes a, the reference is a protein map, the light map is for a microfluidic device with a nominal depth map of 500 μ M, the microfluidic channel map is a nominal map, the abundance of a microfluidic channel map is a 500 μ M map, the abundance of a microfluidic channel map is a microfluidic channel map, the area of a microfluidic channel map is used for a microfluidic channel with a nominal depth map of 500 μ M map, a microfluidic channel map, the microfluidic channel map is a microfluidic channel with a nominal depth map of a microfluidic channel with a microfluidic channel width of a microfluidic channel with.

These results confirm that the expected expression pattern of the tissue sample is repeated using the mapped sequencing data of the multiplex system, and that the multiplex analysis is compatible with immunofluorescence imaging, allowing the determination of cell types based on protein markers and their correlation with gene expression data.

134-retesting Using formalin fixed, Paraffin Embedded (FFPE) samples

One probe pool and 2 device layouts were developed. The probe pool included 134 targets, which represent 69 unique genes as shown in table 1. By sequencing read expression, a few highly expressed genes occupy a large portion of the read, limiting the dynamic range of the assay. This problem is alleviated by attenuating some of the most highly expressed genes in the pool. This is achieved by adding a known proportion of attenuating probes to the active probes. The attenuated probe lacks the 5' phosphate required for the ligation reaction, inhibiting the production of amplification product, thereby reducing the signal of the target. Table 2 shows the attenuation results for the first 5 genes. Before decay they accounted for 73% of the readings and less than 18% thereafter. This strategy can achieve a very high level of multiplex analysis with current sequencing technologies while also achieving a high dynamic range.

Table 1: number of unique targets per gene and Gene List in 134-heavy Probe pool

Table 2: attenuation of first 5 analyte targets

Example 5 elution and preparation of spatially coded probes for next generation sequencing

Using the methods described previously, 134-reprobe pairs were hybridized to FFPE samples using X-position and Y-position linker spatial coding and ligation. In the preparation of the eluent, a hybridization chamber (Agilent) was applied to the coverslip and held in place to form a leak-proof chamber containing the FFPE tissue sample. Using a syringe, the leak-tight chamber was filled with deionized water and heated to 80 ℃ for 30 minutes, after which the eluate was removed using a syringe and transferred to a tube.

The spatially encoded structures were purified by two rounds of positive selection with magnetic beads to separate them from any non-encoded probes. In the first round of purification, the eluate is hybridized to biotinylated capture probes, which comprise a sequence complementary to the sequence spanning the intersection of the X-position linker (address tag) and the 5' ends of the ligated probe pair. The capture probes are then captured by streptavidin-modified magnetic beads, which are then washed on a large scale to remove unbound material. The structure hybridized to the capture probe is then eluted by heating in an elution buffer containing a blocking oligonucleotide complementary to the capture probe. The magnetic beads are separated by the magnetic eluent and transferred to a new container and hybridized with a biotin-labeled capture probe comprising a sequence complementary to the sequence spanning the intersection of the Y-site linker (address tag) and the 3' end of the ligated probe pair. The capture probes, together with the hybridization structures, are subsequently captured on streptavidin-modified magnetic beads and washed.

These beads were transferred directly to a PCR mix containing primers to sequences to effect sequencing of PCR products on an Illumina MiSeq instrument. The primers also included a TruSeq barcode to allow multiplexing of multiple samples in a single sequencing process. A portion of the pcr product was analyzed by gel electrophoresis to check for the presence of amplified spatially encoded structures. The remaining product was purified using QIAGEN PCR purification kit. Finally, the spatially encoded structures were size-selected for purification using a conventional gel electrophoresis apparatus or the Pippen Prep System (Sage Science).

The purified coding structures were sequenced using the Illumina Miseq sequencer and the data used to generate expression profiles.

Example 6 spatially encoded proteins in situ analysis

A highly multiplexed protein assay is performed on a tissue gene chip containing a checkerboard pattern of liver and pancreatic tissue nuclei as described above, in which case a 5-site × 5-site address pattern is used to encode a double assay.A typical immunostaining organization procedure using two primary antibodies, one specific for exocrine pancreatic cells and one specific for liver hepatocytes, is first applied to perform the assay, two antibody-DNA conjugates are used as the secondary antibodies and applied throughout the tissue gene chip.

Example 7 spatially encoded proteins in situ assays

This example describes spatially encoded proteins in an in situ assay. As shown in FIG. 6A, a highly multiplexed protein assay can be performed on samples that preserve the spatial arrangement of cells in the tissue, such as paraffin-embedded or fresh frozen tissue sections fixed to slides. The assay reagent is a protein conjugate (e.g., an antibody) that is recognized by the attachment of a DNA tag that is further encoded by a tag sequence that encodes location or address information (in this example, denoted as X and Y latitudes). The address tags X and Y flank a universal sequence (UP1 and UP2) that can be used as a PCR primer site, a linker for next generation sequencing, or both.

As shown in FIG. 6B, for example, a binder, such as a DNA-labeled antibody probe in this example, is delivered to the entire sample surface in a batch process. The X and Y address labels are then transported to the sample and coupled to the probes such that the probes are encoded by the X and Y address labels according to a spatially defined pattern. In this example, two sets of tags (e.g., a set of 10X address tags, designated X1, X2, X3, … … X10, and a set of 10Y address tags, designated Y1, Y2, Y3, … … Y10) are used in a combinatorial style, and 100 sites in a sample are uniquely identified by the combination of X and Y address tags. For example, a site in the sample shown in fig. XB is uniquely identified as (X9, Y1).

Once the in situ analysis is complete, the assay product is eluted and sequenced. The address tag sequence information analyzes the site at which the analysis is performed, and the probe sequence information identifies the targeted protein. The frequency of a particular assay product at the digital readout can be used to infer the relative abundance of its target sequence in the sample. This information can be correlated with other information, including conventional histological information, and/or transcript abundance obtained by correlated spatially encoded genomic analysis.

Example 8 method for background reduction and signal-to-noise ratio enhancement

This example describes a method for detecting rare variant sequences in mixed nucleic acids. The method may be integrated into the analysis systems and methods of the present invention, for example, to reduce background caused by random errors to improve signal-to-noise (S/N) ratio.

Parallel clonal amplification methods combined with digital sequencing have allowed large-scale variation analysis at 1% resolution (Druley et al, 2009, nat. methods 6:263-65), but not less than 1%. although next generation sequencing technologies can rediscover and allow deeper variation analysis throughout the genome, the combination of factors at different steps in the sequencing process makes it difficult to obtain very low read errors.

FIG. 11A illustrates the concept of rare variation analysis, and FIG. 11B provides a typical configuration of probes that can be used to integrate the analysis of rare mutations into the spatial coding analysis of the present invention. The top panel of fig. 11A shows the target sequence flanked by the linker of interest, which comprises the Illumina linker sequence (labeled a and b) for surface PCR. The target may be obtained from various sources, for example, a PCR amplicon. The linker includes a variable label region (labeled Z). The two strands are shown to illustrate that the Illumina linker is asymmetric. Tagged linkers are used to construct sequencing libraries. On the surface of a flowing cell individual molecules are amplified to form "clusters". The sequence of each target region and its associated tag region is detected. In the final step shown, reads are grouped according to their tag regions, assuming reads with the same tag sequence are of the same origin, assuming z is sufficiently long. These groups were then analyzed to identify rare mutant sequences (the last numbered 4 target was shaded to show that its target sequence was different from that of groups 1-3 compared to the targets represented by groups 1-3). Similar methods for detecting rare mutant sequences have been described in Fu et al, 2011, Proc.Natl.Acad.Sci., 108: 9026-.

Note that although the design as shown in FIG. 11A refers to the Illumina linker and surface amplification method, this method is conventional and can generally be used for library construction methods for other sequencing platforms such as the SO L iD platform (L area technologies), the 454 platform (Roche) and Pacific Biosciences and Ion Torrent.

A model system was established to quantify the improvement in detection limits of standard sequencing using Illumina GAIIx instruments. The model system consisted of a wild-type 100-mer oligonucleotide and a mutant sequence comprising a single site mutation in a unique, wild-type sequence. Synthetic oligonucleotides were cloned into E.coli plasmid vectors and single clones were selected and the sequences verified in order to obtain a structure containing the desired sequence, providing a pure, well-defined sequence structure free of oligonucleotide chemical synthesis errors (typically in the range of 0.3-1%). The 100-mer oligonucleotide of interest is then excised from the plasmid clone by a restriction enzyme. Mutant and wild-type oligonucleotides were quantified and mixed at a ratio of 1:0, 1:20, 1:1000, 1:10,000, 1:100,0001:1,000,000, 0:1 to simulate the presence of rare variations in a wild-type DNA background.

Next, custom linkers including random 10-mer tags were designed and synthesized. Libraries were prepared from defined oligonucleotide mixtures and sequenced using the Illumina GAIIx instrument. The data is analyzed first and no tag information is utilized. This resulted in mutation point detection in only 1:20 samples. The second round of analysis with tags was done with high quality readings in which tag 1/tag 2 groups were retained if tags were grouped together > 99% of the time with > 2 replications of each other. In order for a tag group to be scored as a mutation, at least 90% of the results in the group must match.

Mutant alleles as shown in Table 3 were also successfully detected in the 1:10,000, 1:100,000, 1:1,000,000 samples. Mutant allele frequencies were observed within the expected value of 2, and this difference was attributed to computational and pipetting errors. The ability to observe mutations in the wild type (negative control) sample with the 7.5M tag group was greater than 0.999. Thus, the 1:1,000,000 difference between the spiked samples and the negative control was highly significant.

Table 3: TABLE 3 verification of the ability to detect mutant alleles above the order of 6

The ability to observe mutations with frequency f is the 1- (1-f) ^ # tag, so additional sequencing depth can improve detection capability. The detection limit of the model system is determined only by the sample size and any background contaminants that may be present.

This method is used to distinguish in vitro amplification errors from rare mutations present in the original sample. For example, a simple threshold where the mutation frequency in a tag set must be >0.9 can be used to exclude PCR amplification errors in the analysis. This is based on the observation that: the expected copy part at a particular location contains an error equal to 0.5, provided that the error happens in the first cycle and neglects and the chance of a consecutive PCR error at the same location. In the negative control, no tag passed this standard.

To reduce the background caused by random errors and thereby increase the signal to noise ratio (S/N), such methods can be integrated into methods and detection systems that determine the spatial placement of target abundance, expression or activity. A non-limiting exemplary configuration of a probe that integrates X and Y address labels and a variable label region is shown in fig. 11B.

Example 9 analysis of brain tissue

This example describes the production of an at least 24-fold protein assay plate and confirmation of tissue/cell type specificity by correlation with fluorescent labels and analysis of tissue gene chips.

A panel of 26 antigens were selected, expressed on neurons, astrocytes, oligodendrocytes, microglia or proliferating cells, and antibodies selected against these antigens were commercially available (table 4) in combination with well established staining techniques, which have been successfully used to label different cell types and regions within brain slices (L yck, et al, 2008, J Histochem Cytochem 56,201-21).

The accessibility of antigens is addressed by developing a series of methods for "antigen retrieval" and testing their compatibility with RNA, see MacIntyre, 2001, Br J Biomed sci.58, 190-6; Kap et al, 2011, P L oSOne 6, e 27704; Inoue and Wittbrodt, 2011, P L oS One 6, e19713.

The analysis system was validated by using spatially encoded and conventional IHC fluorescence data and spatially encoded RNA data and applied to brain tissue high-dimensional protein and mRNA data at 32 × 32 sites in human brain tissue-derived sections were generated and compared to published data and brain map data.

Using the methods and detection systems disclosed herein, Allen Brain Atla (www.brain-map. org) can be used to screen target genes for the production of gene expression analysis panels with high information content. The "differential search" tool was used to obtain data from an abundant spatial expression dataset (generated by in situ hybridization) that resulted in the presence of-200 genes in one abundance range in at least one structure/compartment of the brain and/or strong differential expression between different structures/compartments. The selection is reviewed to incorporate any new information or criteria. Probes recognizing this-200 mRNA set were designed and tested for their performance in multiplex assays with reference to on-line gene expression data.

For example, the abundance of at least 24 proteins and 192 mRNAs derived from sections of healthy human brain is analyzed on a 32 × 32 grid of 50 μm pixels.

1. Brain tissue is divided into different substructures: in the anatomical context and at lower levels of multicellular levels;

2. spatial differences indicate different cell types in the tissue (e.g., using a set of proteins/mRNAs known to be specific for a particular cell type)

3. The relationship between mRNA and protein expression of the same gene at different tissue sites.

Table 4: candidate proteins for differentiating brain tissue have been used for immunohistochemistry, and there are commercially available antibodies.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text under the heading, unless otherwise specified.

Citation of the above publications or documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Also, for the purposes of information disclosure, the various figures may depict example structures or other configurations to aid in understanding the features and functionality incorporated into the disclosure. The present disclosure is not limited to the example structures or configurations shown, but may be implemented using a variety of alternate structures and configurations. In addition, while the invention has been described in various exemplary embodiments and implementations, it should be understood that the various features and functions described in one or more embodiments are not limited in their applicability to the particular embodiment described. They may instead be applied, alone or in some combination, to one or more other embodiments of the disclosure, whether or not such embodiments are described, or whether or not such functionality is provided as part of the described embodiments. Thus, the breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments.