Partitioning and processing of analytes and other species

• US 10,150,964 B2
• Filed: 08/31/2017
• Issued: 12/11/2018
• Est. Priority Date: 02/08/2013
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Ness, Kevin, Hardenbol, Paul, Hindson, Christopher, Masquelier, Donald, Jarosz, Mirna, Schnall-Levin, Michael
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/693,374
• Publication Number:
  US 20170362587A1
• Time in Patent Office: 467 Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Claims

1. A method for processing a nucleic acid sample, comprising:

(a) providing a plurality of partitions, wherein a given partition of the plurality of partitions comprises the nucleic acid sample, a plurality of barcode molecules coupled to a bead, and a transposase;

(b) using the transposase to fragment the nucleic acid sample to a plurality of nucleic acid molecules in the given partition; and

(c) using barcode molecules from the plurality of barcode molecules to barcode nucleic acid molecules from the plurality of nucleic acid molecules, thereby providing barcoded nucleic acid molecules,

wherein subsequent to (a), the barcode molecules are released from the bead into the given partition.

2. The method of claim 1, wherein the barcode molecules are released upon application of a stimulus to the given partition, the barcode molecules, or the bead.

3. The method of claim 2, wherein the stimulus is selected from the group consisting of a change in pH, a change in ion concentration, and a reducing agent.

4. The method of claim 1, wherein the bead includes the transposase.

5. The method of claim 1, wherein the bead includes the nucleic acid sample.

6. The method of claim 1, wherein the given partition includes a single cell, and wherein the nucleic acid sample is from the single cell.

7. The method of claim 1, wherein the plurality of partitions is a plurality of droplets.

8. The method of claim 1, wherein the plurality of partitions is a plurality of wells.

9. The method of claim 1, wherein in (a), the nucleic acid sample is provided in the given partition in a predetermined coverage amount.

10. The method of claim 1, further comprising, subsequent to (c), sequencing the barcoded nucleic acid molecules or derivatives thereof.

11. The method of claim 10, wherein the sequencing comprises performing phasing on the barcoded nucleic acid molecules or derivatives thereof.

12. The method of claim 1, further comprising removing the barcoded nucleic acid molecules or derivatives thereof from the given partition.

13. The method of claim 1, further comprising, prior to (a):

providing a cell comprising the nucleic acid sample; and

lysing the cell, thereby generating a lysate.

14. The method of claim 13, wherein the cell is lysed in the given partition.

15. The method of claim 13, wherein the given partition includes a single copy of the nucleic acid sample from the cell.

16. The method of claim 1, wherein (c) is performed in the given partition.

17. The method of claim 1, wherein the nucleic acid sample is a chromosome or portion thereof.

18. The method of claim 1, wherein the plurality of barcode molecules includes at least 1,000 barcode molecules comprising the same nucleic acid sequence.

19. The method of claim 1, wherein the bead is a gel bead.

20. The method of claim 13, wherein the lysate comprises the nucleic acid sample.

21. The method of claim 2, wherein in at least (a), the given partition includes the stimulus.

22. The method of claim 21, wherein the stimulus is a reducing agent.

23. The method of claim 1, wherein each of the plurality of barcode molecules in the given partition has a sequence that is different than sequences of barcode molecules in other partitions of the plurality of partitions.

24. The method of claim 1, wherein the plurality of barcode molecules is coupled to the bead through disulfide bonds.

25. The method of claim 10, wherein the sequencing comprises determining linkage information in sequences generated from the barcoded nucleic acid molecules or derivatives thereof.