Analysis of nucleic acid sequences

Patent metadata

Field	Value
Publication number	US-12312640-B2
Application number	US-202418786391-A
Country	US
Kind code	B2
Filing date	Jul 26, 2024
Priority date	Jun 26, 2014
Publication date	May 27, 2025
Grant date	May 27, 2025

Abstract

The present disclosure relates to methods, compositions and systems for haplotype phasing and copy number variation assays. Included within this disclosure are methods and systems for combining the barcode comprising beads with samples in multiple separate partitions, as well as methods of processing, sequencing and analyzing barcoded samples.

First claim

What is claimed is:

1. A method, comprising:
(a) coupling an antibody to a cell feature of a cell, wherein the antibody is associated with a first barcode molecule comprising a first barcode sequence;
(b) providing a second barcode molecule comprising a second barcode sequence;
(c) from the first barcode molecule and the second barcode molecule, generating a barcoded molecule comprising (i) the first barcode sequence or complement thereof, and (ii) the second barcode sequence or complement thereof;
(d) providing an additional barcode molecule comprising the second barcode sequence; and
(e) from a nucleic acid sequence of the cell and the additional barcode molecule, generating an additional barcoded molecule comprising (i) the nucleic acid sequence or complement thereof, and (ii) the second barcode sequence or complement thereof.

2. The method according to claim 1 , wherein the cell feature of the cell is a protein.

3. The method according to claim 2 , wherein the protein is located on an extracellular surface of the cell.

4. The method according to claim 1 , further comprising:
(f) capturing the barcoded molecule or the additional barcoded molecule with an oligonucleotide molecule comprising a capture sequence.

5. The method according to claim 4 , further comprising capturing the barcoded molecule and the additional barcoded molecule with oligonucleotide molecules that comprise identical capture sequences.

6. The method according to claim 5 , wherein the identical capture sequences comprise poly-thymine (poly-T) sequences.

7. The method according to claim 1 , further comprising sequencing the barcoded molecule or derivative thereof and sequencing the additional barcoded molecule or derivative thereof.

8. The method according to claim 1 , further comprising:
(f) identifying the first barcode sequence and the second barcode sequence; and
(g) using the first barcode sequence and the second barcode sequence identified in (f) to identify the cell feature of the cell and identify the cell, respectively.

9. The method according to claim 1 , wherein (c) and (e) are performed simultaneously.

10. The method according to claim 1 , wherein, prior to (c), the cell is placed in a partition comprising the second barcode sequence.

11. The method according to claim 10 , wherein (c) and (e) are performed in the partition.

12. The method according to claim 10 , wherein the partition comprises a ligase or a reverse transcriptase.

13. The method according to claim 10 , wherein the partition is a droplet.

14. The method according to claim 10 , wherein the partition is a well.

15. The method according to claim 14 , wherein the well is a microwell.

16. The method according to claim 1 , further comprising co-partitioning the cell with a bead, wherein the bead comprises the second barcode molecule and the additional barcode molecule.

17. The method according to claim 16 , wherein the bead is a gel bead.

18. The method according to claim 16 , wherein the bead comprises at least 1,000 barcode molecules comprising the second barcode sequence.

19. The method according to claim 1 , further comprising attaching a functional molecule to the barcoded molecule or derivative thereof.

20. The method according to claim 19 , wherein the functional molecule comprises a biotin tag.

21. The method according to claim 19 , wherein the functional molecule comprises a flow cell attachment sequence.

22. The method according to claim 19 , wherein the functional molecule comprises a unique molecular sequence segment.

23. The method according to claim 1 , further comprising ligating a functional molecule to the additional barcode molecule or derivative thereof.

24. The method according to claim 23 , wherein the functional molecule comprises a biotin tag.

25. The method according to claim 23 , wherein the functional molecule comprises a flow cell attachment sequence.

26. The method according to claim 23 , wherein the functional molecule comprises a unique molecular sequence segment.

27. The method according to claim 1 , wherein the nucleic acid sequence is a ribonucleic acid (RNA) sequence.

28. The method according to claim 27 , wherein the RNA sequence is a messenger RNA (mRNA) sequence.

29. The method according to claim 28 , wherein the mRNA sequence comprises a synthetic nucleic acid sequence.

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• G16B30/20 Primary

  Sequence assembly · CPC title