METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20190203291A1
• Filed: 03/06/2019
• Published: 07/04/2019
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-levin, Michael, Ness, Kevin, Jarosz, Mirna, Saxonv, Serge, Hardenbol, Paul, Bharadwaj, Rajiv, Zheng, Xinying, Belgrader, Phillip
• Granted Patent: US 10,450,607 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/10 the purpose being amplify/increase the copy number of target nucleic acid
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

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 system for nucleic acid processing or analysis, comprising:

a plurality of partitions comprising a plurality of cells, a plurality of antibody-oligonucleotide molecules comprising oligonucleotide sequences, and a plurality of nucleic acid barcode molecules comprising barcode sequences,

wherein antibody-oligonucleotide molecules of said plurality of antibody-oligonucleotide molecules are bound to cells of said plurality of cells, and

wherein a partition of said plurality of partitions comprises;

(i) a single cell of said plurality of cells;

(ii) a subset of antibody-oligonucleotide molecules of said plurality of antibody-oligonucleotide molecules bound to said single cell, wherein said subset of antibody-oligonucleotide molecules comprises an oligonucleotide sequence that differs from oligonucleotide sequences of antibody-oligonucleotide molecules bound to other cells of said plurality of cells; and

(iii) a subset of nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules, wherein said subset of nucleic acid barcode molecules comprises a barcode sequence that differs from barcode sequences of nucleic acid barcode molecules in other partitions of said plurality of partitions.

2. The system of claim 1, wherein said partition comprises a bead, and wherein a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules is attached to said bead.

3. The system of claim 2, wherein said nucleic acid barcode molecule is releasably attached to said bead.

4. The system of claim 2, wherein said nucleic acid barcode molecule is attached to said bead via a disulfide bond.

5. The system of claim 2, wherein, in said partition, said bead comprises an oligonucleotide of an antibody-oligonucleotide molecule of said subset of antibody-oligonucleotide molecules attached thereto.

6. The system of claim 2, wherein said bead is a magnetic bead.

7. The system of claim 6, wherein said magnetic bead is removable from said partition using a magnetic field.

8. The system of claim 2, wherein said bead is a gel bead.

9. The system of claim 2, wherein said bead is a degradable bead.

10. The system of claim 2, wherein said bead comprises polyacrylamide.

11. The system of claim 1, wherein said partition further comprises a reducing agent.

12. The system of claim 1, wherein said partition is a well of a well array.

13. The system of claim 1, wherein said single cell comprises a messenger ribonucleic acid (mRNA), wherein said partition comprises a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules, wherein said nucleic acid barcode molecule is configured to hybridize to said mRNA.

14. The system of claim 1, wherein a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules comprises a unique molecular identification sequence that is unique within said subset of nucleic acid barcode molecules.

15. The system of claim 1, wherein nucleic acid barcode molecules of said subset of nucleic acid barcode molecules comprise a common barcode sequence.

16. The system of claim 1, wherein said plurality of partitions comprises 1,000 partitions.

17. The system of claim 16, wherein said plurality of partitions comprises 10,000 partitions.

18. The system of claim 17, wherein said plurality of partitions comprises 100,000 partitions.

19. The system of claim 18, wherein said plurality of partitions comprises 1,000,000 partitions.

20. The system of claim 1, wherein antibody-oligonucleotide molecules of said plurality of antibody-oligonucleotide molecules configured to bind to a common antigen comprise a common oligonucleotide sequence.

21. The system of claim 1, wherein antibody-oligonucleotide molecules of said plurality of antibody-oligonucleotide molecules configured to bind to different antigens comprise different oligonucleotide sequences.

22. The system of claim 1, wherein different antibody-oligonucleotide molecules of said plurality of antibody-oligonucleotide molecules comprise different oligonucleotide sequences.

23. The system of claim 1, wherein an antibody-oligonucleotide molecule of said subset of antibody-oligonucleotide molecules is bound to surface feature of said single cell.

24. The system of claim 1, wherein an antibody-oligonucleotide molecule of said subset of antibody-oligonucleotide molecules is bound to a protein of said single cell.

25. The system of claim 1, wherein a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules comprises a primer sequence or complement thereof.

26. The system of claim 1, wherein a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules comprises a sequence primer binding sequence or complement thereof.

27. The system of claim 1, wherein a nucleic acid barcode molecule of said subset of nucleic acid barcode molecules comprises an adapter sequence configured to permit attachment to a flow cell of a sequencer or complement thereof.

28. The system of claim 1, wherein said plurality of cells comprises 100 cells.

29. The system of claim 29, wherein said plurality of cells comprises 1,000 cells.

30. The system of claim 30, wherein said plurality of cells comprises 10,000 cells.