Granted Patent B2

US 12,442,045 · App. 18/640,645 · Granted Oct 14, 2025

Inventor: Stephen R. Williams (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6886C12Q1/6874C12Q2600/106C12Q2600/156C12Q2600/158

Abstract

Claims (25)

1. A method of determining a location of an mRNA expressed in an ovarian tissue sample, the method comprising:

(a) contacting the ovarian tissue sample obtained from a subject with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain, wherein a plurality of the capture probes hybridize to a mRNA expressed by a tumor cell in the ovarian tissue sample, and wherein the mRNA expressed by the tumor cell is GJB5 and/or MMP12,

wherein GJB5 mRNA expression is increased in the tumor cell and MMP12 is decreased in the tumor cell as compared to GJB5 mRNA expression and MMP12 mRNA expression in non-tumor cells of the ovarian tissue sample;

(b) hybridizing the mRNAs expressed by the ovarian tissue sample to the plurality of capture probes;

(c) extending the plurality of capture probes, thereby generating a plurality of extended capture probes, wherein a 3′ end of the capture probe uses the mRNA as a template to generate an extended capture probe; and

(d) sequencing (i) all or part of the plurality of extended capture probes, (ii) the spatial barcode or a complement thereof, and (iii) all or part of a sequence corresponding to the mRNA expressed by the ovarian tissue sample, or a complement thereof, and using the determined sequences of (ii) and (iii) to identify the location of the mRNA expressed in the ovarian tissue sample.

2. The method of claim 1 , further comprising comparing expression of the mRNA in the ovarian tissue sample to expression of mRNA in a reference sample.

3. The method of claim 2 , wherein the reference sample comprises a non-cancerous sample from a different subject, a cancerous sample from a different subject, a non-cancerous sample from the subject, or a cancerous sample from the subject.

4. The method of claim 2 , wherein the reference sample is a non-tumor sample from a different subject or a non-tumor sample from the subject.

5. The method of claim 1 , wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, or combinations thereof.

6. The method of claim 1 , wherein the capture domain comprises a poly-uridine sequence or a poly-thymidine sequence.

7. The method of claim 1 , further comprising generating a nucleic acid that is complementary to all or a part of the extended capture probe.

8. The method of claim 1 , wherein the ovarian tissue sample is permeabilized with a permeabilization agent prior to step (b).

9. The method of claim 8 , wherein the permeabilization agent comprises trypsin or a protease.

10. The method of claim 8 , wherein the permeabilization agent comprises pepsin or proteinase K.

11. The method of claim 1 , further comprising amplifying the extended capture probe, or a complement thereof.

12. The method of claim 1 , further comprising determining a location of one or more additional mRNAs expressed by one or more immune cells in the ovarian tissue sample.

13. The method of claim 1 , wherein the mRNA expressed by the tumor cell comprises at least one mutation.

14. The method of claim 13 , wherein the at least one mutation is selected from a substitution, a deletion, a translocation, and an insertion.

15. The method of claim 1 , wherein the ovarian tissue sample is a frozen tissue sample, a fresh tissue sample, or a fixed tissue sample.

16. The method of claim 15 , wherein the fixed tissue sample is a formalin-fixed, paraffin-embedded tissue sample.

17. The method of claim 1 , further comprising, prior to step (a), fixing the ovarian tissue sample.

18. The method of claim 1 , wherein the ovarian tissue sample comprises a fresh-frozen tissue section or a formalin fixed paraffin embedded tissue section.

19. The method of claim 1 , wherein the ovarian tissue sample is stained by hematoxylin and eosin, immunohistochemistry, and/or immunofluorescence.

20. The method of claim 1 , wherein the subject is a human subject.

Assignments (1)

Continuity (4)

References Cited (400)

Cited By (8)

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