Spatially encoded biological assays
The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.
1. A method for determining the presence or abundance of a nucleic acid at a location in a tissue section comprising:
(a) contacting the tissue section with an array comprising a plurality of features, wherein a feature of the plurality of features comprises a capture agent, wherein the capture agent comprises:
(i) a first sequence that binds the nucleic acid, and
(ii) a second sequence comprising a coding tag, wherein a sequence of the coding tag corresponds to a location of the capture agent on the array;
(b) determining the sequence of the coding tag, or a complement thereof, and all or a portion of the sequence of the nucleic acid specifically bound to the capture agent, or a complement thereof, and using the determined sequences to determine presence or abundance of the nucleic acid at a location in the tissue section.
2. The method of claim 1 , wherein the method further comprises, between steps (a) and (b), a step of removing the tissue section from the array.
3. The method of claim 2 , wherein the removing step includes washing the array.
4. The method of claim 1 , wherein the capture agent, when bound to the nucleic acid, can function as a primer.
5. The method of claim 1 , wherein the determining step comprises nucleic acid amplification.
6. The method of claim 1 , wherein the determining step comprises sequencing the coding tag, or a complement thereof, and all or a portion of the sequence of the nucleic acid bound to the capture agent, or a complement thereof.
7. The method of claim 1 , wherein the capture agent further comprises one or both of a primer region and an adaptor region.
8. The method of claim 7 , wherein one or both of the primer region and the adaptor region comprise(s) a universal nucleotide sequence.
9. The method of claim 1 , wherein the nucleic acid is DNA.
10. The method of claim 9 , wherein the DNA is genomic DNA.
11. The method of claim 1 , wherein the nucleic acid is RNA.
12. The method of claim 11 , wherein the RNA is mRNA.
13. The method of claim 1 , wherein the determining step includes generation of a cDNA using the capture agent bound to the nucleic acid as a primer.
14. The method of claim 13 , wherein the generated cDNA comprises the sequence of the coding tag, and a sequence complementary to all or a portion of the sequence of the nucleic acid.
15. The method of claim 13 , wherein the determining step further comprises amplifying the cDNA.
16. The method of claim 1 , wherein the feature of the plurality of features comprises a bead, well, or channel.
17. The method of claim 1 , wherein the capture agent is immobilized directly on a surface of the feature.
18. The method of claim 1 , wherein the capture agent is immobilized indirectly on a surface of the feature.
19. The method of claim 1 , wherein the array comprises a nucleic acid array.
20. The method of claim 19 , wherein the fixed tissue section is a formalin-fixed paraffin embedded (FFPE) tissue section.
21. The method of claim 1 , wherein the array comprises a bead array.
22. The method of claim 1 , wherein the array comprises a chip.
23. The method of claim 1 , wherein the tissue section is a fresh frozen tissue section.
24. The method of claim 1 , wherein the tissue section is a fixed tissue section.
25. The method of claim 1 , wherein the method further comprises imaging the tissue section contacted with the array.
26. The method of claim 25 , wherein the method further comprises aligning an image produced by the imaging step with a fiducial on the array.
27. The method of claim 1 , wherein the method further comprises generating a map of the presence or abundance of the nucleic acid at location(s) in the tissue section.
28. The method of claim 1 , wherein the array comprises greater than 1,000 features.
29. The method of claim 1 , wherein a feature of the plurality of features comprises greater than 50 capture agents, where each of the greater than 50 capture agents comprises a first sequence that binds to a different nucleic acid and a coding tag that corresponds to a location of the capture agent on the array.
30. The method of claim 1 , wherein a feature of the plurality of features comprises greater than 100 capture agents, where each of the greater than 100 capture agents comprises a first sequence that binds to a different nucleic acid and a coding tag that corresponds to a location of the capture agent on the array.