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 substrate, comprising:
a plurality of capture areas, wherein each capture area of the plurality of capture areas is at a different location on the substrate; and
within one or more of the plurality of capture areas, a pair of probes independently immobilized on the substrate, wherein the pair of probes comprises:
a first probe comprising, in a 5′ to 3′ direction, a first primer region comprising an oligonucleotide sequence that is complementary to a first sequencing primer, and a first capture region comprising an oligonucleotide sequence that is complementary to a target nucleic acid sequence; and
a second probe comprising, in a 5′ to 3′ direction, a second primer region comprising an oligonucleotide sequence that is complementary to a second primer different from the first primer, and a ligation region comprising a terminal group that can be ligated to an extension product of the first probe,
wherein at least one of the first and second probes individually, or the first and second probes together, comprise an oligonucleotide sequence that is unique to the capture area within which the pair of probes are immobilized; and
wherein one or more of the plurality of capture areas comprises more than one pair of first and second probes comprising the oligonucleotide sequence that is unique to the capture area within which the more than one pair of first and second probes is independently immobilized.
2. The substrate of claim 1 , wherein the ligation region can be ligated to the extension product of the first probe when the extension product of the first probe comprises at least a portion of a sequence of the target nucleic acid or its complement.
3. The substrate of claim 1 , wherein the first primer region is a universal primer region comprising a constant oligonucleotide sequence.
4. The substrate of claim 1 , wherein the second primer region is a universal primer region comprising a constant oligonucleotide sequence.
5. The substrate of claim 1 , wherein the ligation region comprises a constant oligonucleotide sequence.
6. The substrate of claim 1 , wherein each capture area is spatially separated from the other capture areas on the substrate.
7. The substrate of claim 1 , wherein within each capture area, the second probe is directly immobilized on the substrate or indirectly immobilized on the substrate through a linker.
8. The substrate of claim 1 , wherein within each capture area, the first probe is directly immobilized on the substrate or indirectly immobilized on the substrate through a linker.
9. The substrate of claim 1 , wherein the oligonucleotide sequence of the first capture region is complementary to a single nucleotide polymorphism of the target nucleic acid sequence.
10. The substrate of claim 1 , wherein the oligonucleotide sequence of the first capture region is complementary to a mutation sequence of the target nucleic acid sequence.
11. The substrate of claim 1 , wherein the oligonucleotide sequence of the first capture region is complementary to a genomic DNA sequence of the target nucleic acid sequence.
12. The substrate of claim 1 , wherein the oligonucleotide sequence of the first capture region is complementary to a mRNA sequence of the target nucleic acid sequence.
13. The substrate of claim 1 , wherein the oligonucleotide sequence of the first capture region is complementary to a gene-specific target nucleic acid sequence.
14. The substrate of claim 1 , wherein the substrate comprises a slide or a surface of a flow cell.
15. The substrate of claim 1 , wherein within each capture area of one or more of the plurality of capture areas, the first and second probes are immobilized on a bead that is immobilized on the substrate at the location of the capture area.
16. The substrate of claim 1 , wherein the substrate comprises a fiducial marker.
17. The substrate of claim 1 , wherein one or more of the plurality of capture areas comprises at least 100 pairs of the first and second probes comprising the oligonucleotide sequence that is unique to the capture area within which the pairs of probes are independently immobilized.
18. The substrate of claim 1 , wherein one or more of the plurality of capture areas comprises at least 1000 pairs of the first and second probes comprising the oligonucleotide sequence that is unique to the capture area within which the pairs of probes are independently immobilized.
19. The substrate of claim 1 , wherein:
the target nucleic acid is a first target nucleic acid and the pair of probes is a first pair of probes; and
the substrate comprises, within the one or more of the plurality of capture areas, a second pair of probes immobilized on the substrate, wherein the second pair of probes comprises:
a third probe comprising, in a 5′ to 3′ direction, a third primer region comprising an oligonucleotide sequence that is complementary to a third sequencing primer, and a second capture region comprising an oligonucleotide sequence that is complementary to a second target nucleic acid sequence; and
a fourth probe comprising, in a 5′ to 3′ direction, a fourth primer region comprising an oligonucleotide sequence that is complementary to a fourth primer different from the third primer, and a ligation region comprising a terminal group that can be ligated to an extension product of the third probe,
wherein at least one of the third and fourth probes individually, or the third and fourth probes together, comprise the oligonucleotide sequence that is unique to the capture area within which the second pair of probes is independently immobilized.
20. The substrate of claim 19 , wherein the first and third primer regions are the same.
21. The substrate of claim 19 , wherein the second and fourth primer regions are the same.
22. The substrate of claim 19 , wherein the ligation region of the fourth probe comprises a constant oligonucleotide sequence.
23. The substrate of claim 19 , wherein the oligonucleotide sequences of the first and second capture regions are complementary to different first and second target nucleic acid sequences.
24. The substrate of claim 23 , wherein the oligonucleotide sequences of the first and second capture regions are complementary to different first and second genomic DNA sequences of the first and second target nucleic acid sequences.
25. The substrate of claim 23 , wherein the oligonucleotide sequence of the first capture region is complementary to a DNA sequence of the first target nucleic acid sequence, and the oligonucleotide sequence of the second capture region is complementary to a mRNA sequence of the second target nucleic acid sequence.
26. The substrate of claim 19 , wherein the oligonucleotide sequences of the first and second capture regions are complementary to different first and second mRNA sequences of the first and second target nucleic acid sequences.
27. The substrate of claim 1 , wherein the plurality of capture areas are arranged in a regular array on the substrate.
28. The substrate of claim 1 , wherein the plurality of capture areas comprises at least 1000 capture areas at different locations on the substrate.
29. The substrate of claim 1 , wherein at least one of the first and second probes comprises an adapter region comprising an oligonucleotide sequence that is complementary to a sequencing adapter.