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 a location of a nucleic acid in a tissue sample comprising:
(a) providing sections of the tissue sample;
(b) obtaining an image of a section of the tissue sample in (a) to identify a region of interest in the sections of the tissue sample;
(c) delivering a probe for the nucleic acid to the identified region of interest in a section of the tissue sample in (a), where the probe contains a probe region that selectively binds to the nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered; and
(d) determining all or a portion of a sequence of the probe selectively bound to the nucleic acid or a complement thereof, and using the determined sequence to determine the location of the nucleic acid in the tissue sample.
2. The method of claim 1 , wherein the method further comprises, between steps (c) and (d), a step of separating the probe selectively bound to the nucleic acid from the section of the tissue sample in (c).
3. The method of claim 1 , wherein:
step (b) comprises obtaining an image of a first section of the tissue sample in (a) and using the image to identify a region of interest in a second section of the tissue sample in (a); and
step (c) comprises delivering the probe for the nucleic acid to the identified region of interest in the second section of the tissue sample in (a).
4. The method of claim 1 , wherein the image is obtained using, at least in part, immunohistochemistry or staining.
5. The method of claim 1 , wherein the probe region that specifically binds to the nucleic acid comprises a sequence that is substantially complementary to a portion of the sequence of the nucleic acid.
6. The method of claim 1 , wherein the determining step comprises nucleic acid amplification.
7. The method of claim 1 , wherein the determining step comprises sequencing all or a portion of a sequence of the probe that selectively binds to the nucleic acid or the complement thereof.
8. The method of claim 7 , wherein the probe selectively bound to the nucleic acid can function as a primer.
9. The method of claim 1 , wherein the probe further comprises a universal primer region.
10. The method of claim 1 , wherein the nucleic acid is DNA.
11. The method of claim 10 , wherein the DNA is genomic DNA.
12. The method of claim 1 , wherein the nucleic acid is RNA.
13. The method of claim 12 , wherein the RNA is mRNA.
14. The method of claim 1 , wherein the nucleic acid comprises a single nucleotide polymorphism (SNP) or a mutation.
15. The method of claim 14 , wherein the mutation is a cancer cell mutation.
16. The method of claim 1 , wherein the identified region of interest includes cell types differentiated by immunohistochemistry.
17. The method of claim 1 , wherein the tissue sample comprises diseased tissue.
18. The method of claim 17 , wherein the diseased tissue comprises cancerous tissue, or infected or inflamed tissue.
19. The method of claim 1 , wherein the tissue sample is a frozen tissue sample.
20. The method of claim 1 , wherein the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample.
21. The method of claim 1 , wherein step (c) comprises delivering two different probes for the nucleic acid to the identified region of interest in a section of the tissue sample in (a), and ligating the two probes selectively bound to the nucleic acid to generate a ligation product.
22. The method of claim 21 , wherein the method further comprises between steps (c) and (d), a step of separating the ligation product from the section of the tissue sample in (c).
23. The method of claim 22 , wherein the step of separating comprises the use of an RNase.
24. The method of claim 1 , further comprising producing a map of the location of the nucleic acid in a section of the tissue sample in (a).
25. The method of claim 1 , wherein:
step (b) comprises obtaining an image of a section of the tissue sample in (a) to identify multiple regions of interest in the sections of the tissue sample; and
step (c) comprises delivering the probe for the nucleic acid to the multiple regions of interest in a section of the tissue sample in (a).
26. The method of claim 1 , wherein step (c) comprises delivering greater than 20 probes to the identified region of interest in a section of the tissue sample in (a), where each of the greater than 20 probes contains a probe region that specifically binds to a different nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered.
27. The method of claim 1 , wherein step (c) comprises delivering greater than 50 probes to the identified region of interest in a section of the tissue sample in (a), where each of the greater than 50 probes contains a probe region that specifically binds to a different nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered.
28. The method of claim 1 , wherein step (c) comprises delivering greater than 100 probes to the identified region of interest in a section of the tissue sample in (a), where each of the greater than 100 probes contains a probe region that specifically binds to a different nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered.
29. The method of claim 1 , wherein step (c) comprises delivering greater than 1,000 probes to the identified region of interest in a section of the tissue sample in (a), where each of the greater than 1,000 probes contains a probe region that specifically binds to a different nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered.
30. The method of claim 1 , wherein step (c) comprises delivering greater than 10,000 probes to the identified region of interest in a section of the tissue sample in (a), where each of the greater than 10,000 probes contains a probe region that specifically binds to a different nucleic acid and a coding tag that identifies a site in the identified region of interest to which the probe was delivered.