METHODS AND SYSTEMS FOR DETERMINING SPATIAL PATTERNS OF BIOLOGICAL TARGETS IN A SAMPLE

• US 20160138091A1
• Filed: 06/25/2014
• Published: 05/19/2016
• Est. Priority Date: 06/25/2013
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: CHEE, Mark S., ROUTENBERG, David A.
• Granted Patent: US 9,879,313 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the biological targets comprise proteins, and the methods and assay systems do not depend on imaging techniques for the spatial information of the targets. The present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents; and a decoding scheme providing a readout that is digital in nature.

Claims

1. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises;

(1) a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

(2) an address tag that identifies each of the multiple sites to which the probe is delivered; and

(3) an identity tag that identifies the probe'"'"'s corresponding biological target or target-binding moiety;

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

analyzing the probe bound to the 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 bound to the biological target; and

(2) determining the identities of the identity tag and the address tag of the probe; and

determining a spatial pattern of abundance and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

5. The method of any of claims 1-4, wherein the one or more biological targets are non-nucleic acid molecules.

6. The method of any of claims 1-5, wherein the one or more biological targets are proteins, lipids, carbohydrates, or any combination thereof.

7. The method of any of claims 1-6, wherein there are at least two address tags that identify each of the multiple sites in the sample.

8. The method of any of claims 1-7, wherein spatial patterns of abundance, expression, and/or activity of multiple biological targets are determined in parallel, and the address tag or combination of address tags is the same for each of the multiple biological targets at a given site of the multiple sites in the sample.

9. The method of claim 8, wherein the analyzing step is performed in parallel in the same reaction run.

10. The method of any of claims 1-9, wherein the one or more biological targets include at least one known marker for the sample.

11. The method of claim 10, wherein the known marker is a tissue-specific marker, a cell type marker, a cell lineage marker, a cell morphology marker, a cell cycle marker, a cell death marker, a developmental stage marker, a stem cell or progenitor cell marker, a marker for a differentiated state, an epigenetic marker, a physiological or pathophysiological marker, a marker for a transformed state, a cancer marker, or any combination thereof.

21. The method of any of claims 1-20, wherein the biological target or the target protein comprises an enzyme activity.

22. The method of any of claims 1-21, wherein the target-binding moiety of the probe comprises an antibody or an antigen binding fragment thereof, an aptamer, a small molecule, an enzyme substrate, a putative enzyme substrate, an affinity capture agent, or a combination thereof.

23. The method of any of claims 1-22, wherein the target-binding moiety is conjugated to a polynucleotide comprising the identity tag.

24. The method of any of claims 1-23, wherein the target-binding moiety is conjugated to a polynucleotide capable of specifically hybridizing to a polynucleotide comprising the identity tag.

25. The method of any of claims 1-24, wherein the probe comprises a multiplicity of target-binding moieties capable of binding to the same domain or different domains of the target, or capable of binding to different targets.

26. The method of any of claims 1-25, wherein there are two address tags that identify each of the multiple sites in the sample.

27. The method of any of claims 1-26, wherein two probes for each target are delivered to the sample.

28. The method of any of claims 1-27, wherein the sample is a biological sample selected from the group consisting of a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

29. The method of any of claims 1-28, wherein the address tag comprises an oligonucleotide.

30. The method of any of claims 1-29, wherein the identity tag comprises an oligonucleotide.

31. The method of any of claims 1-30, wherein the analyzing step is performed by nucleic acid sequencing.

32. The method of any of claims 1-31, wherein the analyzing step is performed by high-throughput digital nucleic acid sequencing.

33. The method of any of claims 1-32, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 20.

34. The method of any of claims 1-33, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 50.

35. The method of any of claims 1-34, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 75.

36. The method of any of claims 1-35, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 100.

37. The method of any of claims 1-36, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 1,000.

38. The method of any of claims 1-37, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 10,000.

39. The method of any of claims 1-38, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 100,000.

40. The method of any of claims 1-39, wherein the product of the number of the target(s) being assayed and the number of the multiple sites being assayed in the sample is greater than 1,000,000.

41. The method of any of claims 1-40, wherein at least one hundred thousand probes or probe/address tag conjugates bound to the target(s) are analyzed in parallel.

42. The method of any of claims 1-41, wherein at least five hundred thousand probes or probe/address tag conjugates bound to the target(s) are analyzed in parallel.

43. The method of any of claims 1-42, wherein at least one million probes or probe/address tag conjugates bound to the target(s) are analyzed in parallel.

44. The method of any of claims 1-43, wherein software programmed hardware performs at least two steps of the delivering step(s), the analyzing step(s) and the determining step(s).

45. The method of any of claims 1-44, wherein one or more microfluidic devices are used to perform the delivering step(s).

46. The method of any of claims 1-45, wherein a known percentage of the probe for the biological target, the probe for the target protein, or the probe for the target polynucleotide is an attenuator probe.

47. The method of claim 46, wherein the attenuator probe limits production of an amplifiable product.

48. The method of claim 46 or 47, wherein the attenuator probe lacks a 5′

phosphate.

49. The method of any of claims 1-48, wherein the address tag is coupled to the probe by ligation or by extension or any combination thereof.

50. The method of any of claims 1-49, further comprising constructing a 3-dimensional pattern of abundance, expression, and/or activity of each target from spatial patterns of abundance, expression, and/or activity of each target of multiple samples.

51. The method of claim 50, wherein the multiple samples are consecutive tissue sections of a 3-dimensional tissue sample.

2. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises;

(1) a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

(2) an address tag having a sequence that identifies each of the multiple sites to which the probe is delivered; and

(3) an identity tag having a sequence that identifies the probe'"'"'s corresponding biological target or target-binding moiety;

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

analyzing the probe bound to the one or more biological targets by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis.

3. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises;

(1) a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target; and

(2) an identity tag that identifies the probe'"'"'s corresponding biological target or target-binding moiety;

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

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the probe/address tag conjugate bound to the 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 identities of the identity tag and the address tag of the probe/address tag conjugate; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

4. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises;

(1) a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target; and

(2) an identity tag having a sequence that identifies the probe'"'"'s corresponding biological target or target-binding moiety;

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

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the probe/address tag conjugate bound to the one or more biological targets by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis.

12. A method of determining a spatial pattern of abundance, expression, and/or activity of a target protein across multiple sites in a sample, comprising:

delivering a probe for a target protein to multiple sites in a 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 multiple sites to which the probe is delivered; and

(3) an identity tag that identifies the target protein or the target-binding moiety;

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

analyzing the probe bound to the target protein, the analysis comprising;

(1) determining 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 identities of the identity tag and the first address tag of the probe for the target protein; and

determining a spatial pattern of abundance, expression, and/or activity of the target protein across the multiple sites in the sample based on the analysis.

13. The method of claim 12, further comprising:

delivering a probe for a target polynucleotide to each of the multiple sites in the sample, wherein the probe for the target polynucleotide comprises;

(1) a sequence that hybridizes to and identifies the target polynucleotide;

(2) a second address tag that identifies each of the multiple sites to which the probe for the target polynucleotide is delivered;

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

analyzing the probe bound to the target polynucleotide, the analysis comprising;

(1) determining abundance, expression, and/or activity of the target polynucleotide by assessing the amount of the probe bound to the target polynucleotide; and

(2) determining the identities of the sequence that hybridizes to and identifies the target polynucleotide and the second address tag of the probe for the target polynucleotide; and

determining a spatial pattern of abundance, expression, and/or activity of the target polynucleotide across the multiple sites in the sample based on the analysis of the probe bound to the target polynucleotide at each of the multiple sites in the sample.

16. The method of claim 13 or 15, wherein the target polynucleotide or the complement thereof encodes all or a portion of the target protein.

17. The method of claim 13 or 15, wherein 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 polynucleotide are performed in parallel in the same reaction run.

18. The method of any of claims 13, 15, 16, and 17, wherein the first address tag and the second address tag are the same for a given site of the multiple sites in the sample.

19. The method of any of claims 13, 15, 16, and 17, wherein the first address tag and the second address tag are different for a given site of the multiple sites in the sample.

20. The method of any of claims 13, 15, 16, 17, 18, and 19, further comprising associating abundance, expression, and/or activity of the target protein to abundance, expression, and/or activity of the target polynucleotide at each of the multiple sites in the sample.

14. A method of determining a spatial pattern of abundance, expression, and/or activity of a target protein across multiple sites in a sample, comprising:

delivering a probe for a target protein to multiple sites in the sample, wherein the probe comprises;

(1) a target-binding moiety capable of binding to the target protein; and

(2) an identity tag that identifies the target protein or the protein-binding moiety;

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

delivering a first address tag to each of the multiple sites in the sample, wherein the first address tag is to be coupled to the probe bound to the target protein and identifies the site to which it is delivered;

analyzing the probe/first address tag conjugate bound to the target protein, the analysis comprising;

(1) determining abundance, expression, and/or activity of the target protein by assessing the amount of the probe/first address tag conjugate bound to the target protein; and

(2) determining the identities of the identity tag and the first address tag of the probe/first address tag conjugate; and

determining a spatial pattern of abundance, expression, and/or activity of the target protein across the multiple sites in the sample based on the analysis.

15. The method of claim 14, further comprising:

delivering a probe for a target polynucleotide to each of the multiple sites in the sample, wherein the probe for the target polynucleotide comprises a sequence that hybridizes to and identifies the target polynucleotide;

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

delivering a second address tag to each of the multiple sites in the sample, wherein the second address tag is to be coupled to the probe bound to the target polynucleotide and identifies the site to which it is delivered;

analyzing the probe/second address tag conjugate bound to the target polynucleotide, the analysis comprising;

(1) determining abundance, expression, and/or activity of the target polynucleotide by assessing the amount of the probe/second address tag conjugate bound to the target polynucleotide; and

(2) determining the identities of the sequence that hybridizes to and identifies the target polynucleotide and the second address tag of the probe/second address tag conjugate; and

determining a spatial pattern of abundance, expression, and/or activity of the target polynucleotide across the multiple sites in the sample based on the analysis of the probe/second address tag conjugate bound to the target polynucleotide at each of the multiple sites in the sample.

52. A system for determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

a first module for delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises;

(1) a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target; and

(2) an identity tag that identifies the probe'"'"'s corresponding biological target or target-binding moiety;

a second module for delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the probe bound to the biological target and identifies the site to which the address tag is delivered;

a third module for analyzing the probe/address tag conjugate 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 identities of the identity tag and the address tag of the probe/address tag conjugate; and

a fourth module for determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the system does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

53. The system of claim 52, wherein the second module comprises one or more microfluidic devices for delivering the address tags.

54. The system of claim 53, wherein the one or more microfluidic devices comprise a first set of multiple addressing channels, each delivering a different first address tag to the sample.

55. The system of claim 54, wherein the one or more microfluidic devices further comprise a second set of multiple addressing channels, each delivering a different second address tag to the sample.

56. The method of claim 55, wherein the multiple sites in the sample are chosen by the first and second set of multiple addressing channels cooperatively delivering the first address tags and the second address tags, respectively, to each of the multiple sites, each site identified by a different combination of first and second address tags.

57. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

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

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe and comprises an address tag that identifies each of the multiple sites to which the at least one adaptor is delivered, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

analyzing the at least one adaptor and the probe bound to the 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 at least one adaptor bound to the probe bound to the biological target; and

(2) determining the identities of the identity tag, and the address tag of the at least one adaptor; and

determining a spatial pattern of abundance and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

61. The method of any one of claims 57-60, wherein at least two adaptors are delivered to each of the multiple sites in the sample, wherein the at least two adaptors each specifically binds to one probe that specifically binds to the biological target.

62. The method of claim 61, wherein the at least two adaptors are joined.

63. The method of claim 62, wherein the at least two adaptors are ligated by using a portion of the probe sequence as a splint.

64. The method of any one of claims 57-63, wherein the probe for each of the one or more biological targets comprises an affinity agent for the biological target and an oligonucleotide, and the adaptor comprises an oligonucleotide.

58. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

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

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe and comprises an address tag that identifies each of the multiple sites to which the at least one adaptor is delivered, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

analyzing the at least one adaptor and the probe bound to the one or more biological targets by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

59. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

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

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the at least one adaptor bound to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the adaptor/address tag conjugate, 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 adaptor/address tag conjugate bound to the probe bound to the biological target; and

(2) determining the identities of the identity tag, and the address tag of the adaptor/address tag conjugate; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis,

wherein the method does not depend on an imaging technique for determining spatial information of the one or more biological targets in the sample.

60. A method of determining a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample, comprising:

delivering a probe for each of one or more biological targets to multiple sites in a sample, wherein each probe comprises a target-binding moiety capable of binding to the probe'"'"'s corresponding biological target;

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

delivering at least one adaptor to the multiple sites in the sample, wherein the at least one adaptor specifically binds to the probe, wherein the probe and/or the adaptor comprises an identity tag that identifies the probe'"'"'s and/or adaptor'"'"'s corresponding biological target or target-binding moiety;

delivering an address tag to each of the multiple sites in the sample, wherein the address tag is to be coupled to the at least one adaptor bound to the probe bound to the biological target and identifies the site to which the address tag is delivered;

analyzing the adaptor/address tag conjugate by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of each of the one or more biological targets, the sequencing product comprising all or a portion of the address tag sequence and all or a portion of the identity tag sequence; and

determining a spatial pattern of abundance, expression, and/or activity of the one or more biological targets across the multiple sites in the sample based on the analysis.