Methods and systems for determining spatial patterns of biological targets in a sample
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.
1. A method of determining a location of a target polynucleotide in a sample comprising:
(a) attaching the sample affixed to a support to a first microfluidic device having multiple first addressing channels, wherein a first addressing channel identifies a first area in the sample;
(b) delivering a first probe through the first addressing channel to the first area in the sample, wherein the first probe comprises (i) a sequence substantially complementary to at least a portion of the target polynucleotide, (ii) a first address tag that identifies the first area in the sample, and (iii) a first ligation region;
(c) attaching the sample affixed to the support to a second microfluidic device having multiple second addressing channels, wherein a second addressing channel identifies in the sample a second area that intersects with the first area; and
(d) delivering a second probe through the second addressing channel to the second area in the sample, wherein the second probe comprises: (i) a second address tag that identifies the second area in the sample and (ii) a second ligation region, wherein the second probe is coupled to the first probe through ligation between the first and second ligation regions at an intersection between the first area and the second area, and the first and second address tags are used to identify the location of the target polynucleotide at the intersection in the sample.
2. The method of claim 1 , wherein the multiple first addressing channels are substantially parallel to each other and the multiple second addressing channels are substantially parallel to each other.
3. The method of claim 1 , wherein the multiple first addressing channels and/or the multiple second addressing channels is n addressing channels, wherein n is an integer between 20 and 1000.
4. The method of claim 1 , wherein the width of each of the multiple first addressing channels and/or each of the multiple second addressing channels is about 1 μm to about 500 μm.
5. The method of claim 1 , wherein the depth of each of the multiple first addressing channels and/or each of the multiple second addressing channels is about 1 μm to about 500 μm.
6. The method of claim 1 , wherein the distance between each of the multiple first addressing channels and/or between each of the multiple second addressing channels is about 1 μm to about 2.0 mm.
7. The method of claim 1 , wherein the second area forms an angle with the first area at the intersection, and wherein the angle is about 10 degrees to about 90 degrees.
8. The method of claim 1 , further comprising, between steps (b) and (c), allowing the first probe to hybridize with the target polynucleotide, and extending the first probe using the target polynucleotide as a template.
9. The method of claim 1 , wherein the second probe further comprises a variable tag region, a sequencing adaptor, or a combination thereof.
10. The method of claim 1 , wherein the second probe is coupled with the first probe by extension followed by ligation.
11. The method of claim 1 , wherein the sample is a freshly isolated tissue sample, a fixed tissue sample, a frozen tissue sample, an embedded tissue sample, a processed tissue sample, or a combination thereof.
12. The method of claim 1 , wherein the method further comprises determining the sequence of the first and second address tags by nucleic acid sequencing or high-throughput sequencing.
13. The method of claim 1 , wherein the target polynucleotide is an RNA.
14. The method of claim 13 , wherein the RNA is an mRNA.
15. A method of determining a location of a target protein in a sample comprising:
(a) delivering a first probe to the sample, wherein the first probe comprises: (i) a binding moiety that specifically binds to the target protein, and (ii) an oligonucleotide conjugated to the binding moiety;
(b) attaching the sample to a first microfluidic device having multiple first addressing channels, wherein a first addressing channel identifies a first area in the sample;
(c) delivering a second probe through the first addressing channel to the first area in the sample, wherein the second probe comprises: (i) a sequence substantially complementary to a portion of the sequence of the oligonucleotide of step (a), (ii) a sequence substantially complementary to a portion of a target polynucleotide, (iii) a first address tag that identifies the first area in the sample, and (iv) a first ligation region;
(d) attaching the sample from step (c) to a second microfluidic device having multiple second addressing channels, wherein a second addressing channel identifies in the sample a second area that intersects with the first area; and
(e) delivering a third probe through the second addressing channel to the second area in the sample, wherein the third probe comprises (i) a second address tag that identifies the second area in the sample, and (ii) a second ligation region, wherein the second probe is coupled with the third probe through ligation between the first and second ligation regions at an intersection between the first area and the second area, and the first and second address tags are used to identify the location of the target protein at the intersection in the sample.
16. The method of claim 15 , further comprising, between steps (c) and (d), extending the second probe using the oligonucleotide of step (a) as a template.
17. The method of claim 15 , wherein the third probe further comprises a variable tag region, a sequencing adaptor, or a combination thereof.
18. The method of claim 15 , wherein the target polynucleotide or a complement thereof encodes all or a portion of the target protein.
19. The method of claim 15 , wherein the target protein comprises an intracellular protein.
20. The method of claim 15 , wherein the target protein comprises an extracellular protein.
21. The method of claim 15 , wherein the binding moiety comprises an antibody or an antigen binding fragment thereof, an aptamer, a small molecule, an enzyme substrate, an affinity capture agent, or a combination thereof.
22. A method of determining a location of a target protein in a sample, comprising:
(a) delivering a first probe for the target protein to multiple locations in the sample, the first probe comprising (i) a binding moiety capable of specifically binding to the target protein, and (ii) an oligonucleotide conjugated to the binding moiety;
(b) delivering a second probe to a first location in the sample, wherein the second probe comprises: (i) a sequence substantially complementary to a portion of the sequence of the oligonucleotide of step (a), (ii) a sequence substantially complementary to at least a portion of a target polynucleotide; (iii) a first address tag that identifies the first location in the sample, and (iv) a first ligation region; and
(c) delivering a third probe to a second location in the sample, wherein the third probe comprises (i) a second address tag that identifies the second location in the sample to which the third probe is delivered, and (ii) a second ligation region, wherein the second probe is coupled with the third probe through ligation between the first and second ligation regions at an intersection between the first and second locations in the sample, and the first and second address tags are used to determine the location of the target protein at the intersection.
23. The method of claim 22 , comprising, between steps (b) and (c), extending the second probe using the oligonucleotide of step (a) as a template.
24. The method of claim 22 , wherein the third probe further comprises a variable tag region, a sequencing adaptor, or a combination thereof.
25. The method of claim 22 , wherein the target polynucleotide or a complement thereof encodes all or a portion of the target protein.
26. The method of claim 22 , wherein the target protein comprises an intracellular protein.
27. The method of claim 22 , wherein the target protein comprises an extracellular protein.
28. The method of claim 22 , wherein the binding moiety comprises an antibody or an antigen binding fragment thereof, an aptamer, a small molecule, an enzyme substrate, an affinity capture agent, or a combination thereof.