US-12624475-B1 - Capturing genetic targets using a hybridization approach

Abstract

Provided herein are methods of determining a location of an analyte using hybridization as a method for enhancing detection of the analyte.

Inventors

• Andrew Scott Kohlway
• Francesca Meschi
• Jennifer Chew
• Joseph Glenn Arthur
• Nigel DELANEY
• Zachary Bent
• Katherine Pfeiffer
• Andrew John Hill
• Luigi Jhon Alvarado Martinez

Assignees

• 10X GENOMICS, INC.

Dates

Publication Date

20260512

Application Date

20220822

Claims (20)

1. 1 . A method for enriching an analyte in a biological sample, which is a tissue sample, wherein the analyte is a nucleic acid which is an RNA molecule, the method comprising: (a) contacting the biological sample with a substrate comprising a plurality of attached capture probes, wherein a capture probe of the plurality of attached capture probes comprises (i) a spatial barcode and (ii) a capture domain that hybridizes to a sequence present in the analyte; (b) hybridizing the capture probe to the analyte; (c) extending a 3′ end of the capture probe using the analyte that is bound to the capture probe as a template to generate an extended capture probe; (d) contacting a plurality of nucleic acids with a plurality of bait oligonucleotides, wherein: the plurality of nucleic acids comprises the extended capture probe comprising (i) the spatial barcode, or a complement thereof, wherein the spatial barcode is a nucleic acid sequence that provides information as to the location or position of an analyte with in the biological sample, and (ii) all or a portion of a sequence of the analyte, or a complement thereof; and a bait oligonucleotide of the plurality of bait oligonucleotides comprises: (i) a bait capture domain that hybridizes to a particular exon in the sequence of the analyte from the biological sample, or a complement thereof, and (ii) a molecular tag; (e) capturing a complex of the bait oligonucleotide bound to the extended capture probe using a second substrate comprising an agent that binds to the molecular tag; and (f) isolating the complex of the bait oligonucleotide bound to the extended capture probe, thereby enriching the analyte in the biological sample.
2. 2 . The method of claim 1 , further comprising determining (i) the sequence of the spatial barcode or the complement thereof, and (ii) all or a portion of the sequence of the extended capture probe, and using the determined sequences of (i) and (ii), thereby identifying the to identify the location of the analyte in the biological sample.
3. 3 . The method of claim 1 , further comprising amplifying the extended capture probe.
4. 4 . The method of claim 1 , wherein the extended capture probe is released from the substrate.
5. 5 . The method of claim 1 , wherein step (d) and step (e) are performed at substantially the same time.
6. 6 . The method of claim 1 , wherein the bait capture domain of the bait oligonucleotide comprises a total of about 10 nucleotides to about 300 nucleotides.
7. 7 . The method of claim 1 , wherein the bait capture domain of the bait oligonucleotide comprises a total of about 120 nucleotides.
8. 8 . The method of claim 1 , wherein the bait capture domain of the bait oligonucleotide hybridizes to a 3′ portion, a 5′ portion, an intron, an exon, an untranslated 3′ region, or an untranslated 5′ region of the sequence of the analyte.
9. 9 . The method of claim 1 , wherein the molecular tag comprises a protein, a small molecule, a nucleic acid, or a carbohydrate.
10. 10 . The method of claim 1 , wherein the molecular tag is streptavidin, avidin, biotin, or a fluorophore.
11. 11 . The method of claim 1 , wherein the agent comprises a protein, a nucleic acid, or a small molecule.
12. 12 . The method of claim 1 , wherein the molecular tag is biotin and the agent is avidin or streptavidin.
13. 13 . The method of claim 1 , wherein the agent is attached to the second substrate.
14. 14 . The method of claim 13 , wherein the second substrate is a bead, a well, or a slide.
15. 15 . The method of claim 1 , wherein the biological sample is a tissue sample.
16. 16 . The method of claim 1 , wherein the tissue sample is a formalin-fixed, paraffin-embedded tissue sample.
17. 17 . The method of claim 1 , wherein the tissue sample is a fresh tissue sample or a frozen tissue sample.
18. 18 . The method of claim 1 , wherein the biological sample was previously stained using a hematoxylin and eosin stain, immunofluorescence, or immunohistochemistry.
19. 19 . The method of claim 18 , further comprising imaging the biological sample.
20. 20 . The method of claim 2 , wherein the determining step comprises sequencing (i) the sequence of the spatial barcode or the complement thereof, and (ii) all or a portion of the extended capture probe.

Description

CROSS-REFERENCE TO RELATED APPLICATION Pursuant to 35 U.S.C. § 119(e), this application is a continuation of International Application PCT/US2021/018795, with an international filing date of Feb. 19, 2021, which claims priority to U.S. Provisional Patent Application No. 62/979,652, filed Feb. 21, 2020; U.S. Provisional Patent Application No. 62/980,124, filed Feb. 21, 2020; and U.S. Provisional Patent Application No. 63/077,019, filed Sep. 11, 2020. The contents of the foregoing applications are incorporated herein by reference in its entirety. BACKGROUND Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte abundance (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and cross-talk with other cells in the tissue. Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide a lot of analyte data for single cells, but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample). Whole exome sequencing provides coverage for each transcripts in a sample. There is a need in the art for a transcriptome-specific method for the high-fidelity enrichment of nucleic acid molecules for targeted sequencing while reducing cost, maximizing efficiency, and minimizing redundancy. SUMMARY Disclosed herein is a method for identifying abundance and location of an analyte in a biological sample, the method comprising: (a) contacting a plurality of nucleic acids with a plurality of bait oligonucleotides, in some embodiments, the plurality of nucleic acids comprises an extended nucleic acid comprising (i) a spatial barcode or a complement thereof, and (ii) all or a portion of a sequence of the analyte, an analyte derivative, or a complement thereof; and a bait oligonucleotide of the plurality of bait oligonucleotides comprises: (i) a capture domain that hybridizes to all or a portion of the sequence of the analyte, the analyte derivative, or a complement thereof, and (ii) a molecular tag; (b) capturing the bait oligonucleotide bound to the extended nucleic acid using a substrate comprising an agent that binds to the molecular tag; and (c) determining (i) all or a portion of the sequence of the spatial barcode or the complement thereof, and (ii) all or a portion of the sequence of the extended nucleic acid, and using the determined sequences of (i) and (ii) to identify the abundance and the location of the analyte in the biological sample. In some embodiments, the analyte is a nucleic acid. In some embodiments, the method further comprises generating the plurality of nucleic acids, which comprises: (a) contacting the biological sample with a substrate comprising a plurality of attached capture probes, in some embodiments, a capture probe of the plurality comprises (i) the spatial barcode and (ii) a capture domain that binds to a sequence present in the nucleic acid; (b) hybridizing the capture probe to the nucleic acid; (c) extending a 3′ end of the capture probe using the nucleic acid that is bound to the capture domain as a template to generate an extended capture probe; and (d) amplifying the extended capture probe to produce the extended nucleic acid. In some embodiments, the extended nucleic acid is released from the extended capture probe. In some embodiments, the analyte is a protein. In some embodiments, the analyte derivative is an oligonucleotide comprising an analyte binding moiety barcode, and an analyte capture sequence. In some embodiments, the method further comprises generating the plurality of nucleic acids, the method comprising: (a) contacting a plurality of analyte capture agents to the biological sample, in some embodiments, an analyte capture agent of the plurality of the analyte capture agents comprises (i) an analyte binding moiety that binds to the protein and (ii) the oligonucleotide comprising the analyte binding moiety barcode and the analyte capture sequence; (b) contacting the plurality of analyte capture agents to a substrate comprises a plurality of capture probes, in some embodiments, a capture probe of the plurality comprises the spatial barcode and a capture domain, in some embodiments, the capture domain binds to the analyte capture sequence; (c) extending a 3′ end of the capture probe using the analyte binding moiety barcode as a template to generate an extended capture probe; and (d) amplifying the extended capture probe to produce the extended nucleic acid. In some embodiments, the extended nucleic acid is released from the extended capture probe. In som