Granted Patent B2

US 12,421,539 · App. 18/520,514 · Granted Sep 23, 2025

Inventors: Xinying Zheng (San Jose, CA); Serge Saxonov (Oakland, CA); Michael Schnall-Levin (San Francisco, CA); Kevin Ness (Pleasanton, CA); Rajiv Bharadwaj (Pleasanton, CA)

Assignee: 10x Genomics, Inc.

C12Q1/6809C12Q1/6806C12Q1/6834C12Q1/6869

Abstract

Claims (25)

1. A method of analyzing nucleic acids in a formalin fixed paraffin embedded (FFPE) tissue sample while maintaining spatial context, the method comprising:

(a) providing a FFPE tissue sample comprising nucleic acids;

(b) applying a library of tags to the tissue sample such that different geographical regions of the tissue sample receive different tags or different concentrations of tags;

(c) following (b), separating portions of the tissue sample into discrete partitions such that portions of the library of tags and portions of the nucleic acids are also separated into the discrete partitions;

(d) obtaining sequence information from the nucleic acids, and

(e) identifying tags or concentrations of tags in the discrete partitions, thereby analyzing said nucleic acids while maintaining spatial context.

2. The method of claim 1 , wherein the applying step (b) results in different concentrations of tags in different spatial locations in the tissue sample.

3. The method of claim 2 , wherein the identifying step (e) comprises determining the concentrations of tags in the discrete partitions.

4. The method of claim 1 , wherein the tags are applied to the tissue sample in step (b) such that different regions of the tissue sample receive different tags.

5. The method of claim 4 , wherein identifying in (e) comprises identifying sequences of the tags in each of the discrete partitions.

6. The method of claim 1 , wherein the tags comprise oligonucleotide barcodes.

7. The method of claim 1 , wherein the tissue sample is deparaffinized prior to the (d) obtaining.

8. The method of claim 1 , wherein the nucleic acids comprise RNA.

9. The method of claim 1 , wherein the amount of nucleic acid in each discrete partition is less than 10 ng/ml, optionally wherein the amount of nucleic acid in each discrete partition is less than 1 ng/ml.

10. The method of claim 1 , wherein the tissue sample is a tumor sample.

11. The method of claim 1 , wherein step (d) comprises a sequencing reaction selected from the group consisting of short read-length sequencing reactions and long read-length sequencing reactions, optionally wherein the sequencing reaction is a short read, high accuracy sequencing reaction.

12. The method of claim 1 , wherein a plurality of barcoded nucleic acid fragments are produced in each discrete partition prior to (d); optionally wherein:

each of the barcoded nucleic acid fragments in each discrete partition comprises a copy of a fragment of the portion of nucleic acids in the partition and a partition-specific barcode;

each of the barcoded nucleic acid fragments in a discrete partition comprise the same partition-specific barcode; or

the sequence information obtained in (d) is obtained by sequencing the plurality of barcoded nucleic acid fragments in each partition.

13. The method of claim 1 , wherein each partition further comprises a bead, the bead comprising a plurality of partition-specific barcode oligonucleotides releaseably attached to said bead.

14. The method of claim 13 , wherein the partition-specific barcode oligonucleotides each comprise a barcode region and a random n-mer region; optionally wherein:

the partition-specific barcode oligonucleotides in each discrete partition comprises the same barcode region and a different random n-mer region; or

the partition-specific barcode oligonucleotides in a discrete partition are identical and the partition-specific barcodes between discrete partitions are different.

15. The method of claim 13 , wherein the bead is a gel bead.

Assignments (1)

Continuity (5)

References Cited (400)