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 of determining the presence or abundance of a plurality of biological molecules in a location of interest in a biological sample comprising:
(a) contacting the biological sample with a first and a second probe for each of the plurality of biological molecules, wherein a first and second probe for each biological molecule each comprise a sequence that is complementary to a sequence in the biological molecule, and when the first and second probes are hybridized to the biological molecule, the first and second probes are capable of being ligated together;
(b) imaging the biological sample to identify the location of interest;
(c) ligating first and second probes that are hybridized to a biological molecule to generate a ligation product(s) having a sequence;
(d) separating the ligation product(s) from the location of interest in the biological sample; and
(e) determining the sequence of the separated ligation product(s), thereby determining the presence or abundance of the plurality of biological molecules at the location of interest in the biological sample,
wherein step (b) is performed before step (d).
2. The method of claim 1 , further comprising, after step (a), washing unhybridized first and second probes from the biological sample.
3. The method of claim 1 , wherein step (b) is performed before step (a), between step (a) and step (c), or between step (c) and step (d).
4. The method of claim 1 , wherein the plurality of biological molecules is RNA or DNA.
5. The method of claim 1 , wherein the plurality of biological molecules represents a transcriptional pattern in the location of interest of the biological sample.
6. The method of claim 1 , wherein the support is a slide or a culture dish.
7. The method of claim 1 , wherein the biological sample is affixed to a support.
8. The method of claim 1 , wherein the biological sample affixed to the support is a paraformalin-fixed, paraffin-embedded (FFPE) or frozen sample.
9. The method of claim 1 , wherein the imaging is performed using microscopy.
10. The method of claim 1 , determining the sequence of the separated ligation product(s) includes the performance of high-throughput, next-generation sequencing.
11. The method of claim 1 , further comprising a step of amplifying the ligation product(s) between the separating step and the determining step.
12. A method of determining the presence or abundance of a plurality of biological molecules in a location of interest in a biological sample comprising:
(a) imaging a biological sample to identify the location of interest;
(b) contacting the location of interest in the biological sample with a first and a second probe for each of the plurality of biological molecules, wherein a first and second probe for each biological molecule each comprise a sequence that is complementary to a sequence in the biological molecule, and when the first and second probes are hybridized to the biological molecule, the first and second probes are capable of being ligated together;
(c) ligating first and second probes that are hybridized to a biological molecule in the location of interest in the biological sample to generate a ligation product(s) having a sequence;
(d) separating the ligation product(s) from the location of interest in the biological sample; and
(e) determining the sequence of the separated ligation product(s), thereby determining the presence or abundance of the plurality of biological molecules in the location of interest in the biological sample.
13. The method of claim 12 , further comprising, after step (b), washing unhybridized first and second probes from the biological sample.
14. The method claim 12 , wherein the plurality of biological molecules is RNA or DNA.
15. The method of claim 14 , wherein the plurality of biological molecules represents a transcriptional pattern in the location of interest of the biological sample.
16. The method of claim 12 , wherein the support is a slide or a culture dish.
17. The method of claim 12 , wherein the biological sample is affixed to a support.
18. The method of claim 12 , wherein the biological sample affixed to the support is a paraformalin-fixed, paraffin-embedded (FFPE) or frozen sample.
19. The method of claim 12 , wherein the imaging is performed using microscopy.
20. The method of claim 12 , determining the sequence of the separated ligation product(s) includes the performance of high-throughput, next-generation sequencing.
21. The method of claim 12 , further comprising a step of amplifying the ligation product(s) between the separating step and the determining step.
22. A method of determining the presence or abundance of a plurality of biological molecules in a location of interest in a biological sample comprising:
(a) imaging a biological sample to identify the location of interest;
(b) contacting the location of interest in the biological sample with a first and a second probe for each of the plurality of biological molecules, wherein a first and second probe for each biological molecule each comprise a sequence that is complementary to a sequence in the biological molecule, and when the first and second probes are hybridized to the biological molecule, the first and second probes are capable of being ligated together;
(c) ligating first and second probes that are hybridized to a biological molecule in the biological sample to generate a ligation product(s) having a sequence;
(d) separating the ligation product(s) from the biological sample;
(e) determining the sequence of the separated ligation product(s), thereby determining the presence or abundance of the plurality of biological molecules in the location of interest in the biological sample.
23. The method of claim 22 , further comprising, after step (b), washing unhybridized first and second probes from the biological sample.
24. The method claim 22 , wherein the plurality of biological molecules is RNA or DNA.
25. The method of claim 22 , wherein the plurality of biological molecules represents a transcriptional pattern in the location of interest of the biological sample.
26. The method of claim 22 , wherein the support is a slide or a culture dish.
27. The method of claim 22 , wherein the biological sample is a frozen sample or a paraformalin-fixed, paraffin-embedded (FFPE) sample.
28. The method of claim 22 , determining the sequence of the separated ligation product(s) includes the performance of high-throughput, next-generation sequencing.
29. The method of claim 22 , further comprising a step of amplifying the ligation product(s) between the separating step and the determining step.