Methods and Systems for Processing Polynucleotides

• US 20190032129A1
• Filed: 09/27/2018
• Published: 01/31/2019
• Est. Priority Date: 06/26/2014
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Saxonov, Serge, Bharadwaj, Rajiv
• Granted Patent: US 10,457,986 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• 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 2565/629 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 method for processing or analyzing one or more components from a cell, comprising:

(a) providing a cell bead and a barcode bead, wherein (i) said cell bead comprises said one or more components of said cell, wherein said one or more components comprise a nucleic acid molecule, and (ii) said barcode bead comprises a plurality of nucleic acid barcode molecules for barcoding said nucleic acid molecule; and

(b) partitioning said cell bead and said barcode bead into a partition, wherein upon partitioning, said partition comprises said cell bead and said barcode bead.

2. The method of claim 1, further comprising performing one or more reactions on said nucleic acid molecule.

3. The method of claim 2, wherein said one or more reactions comprise a reaction selected from the group consisting of nucleic acid modification, nucleic acid amplification, nucleic acid insertion, nucleic acid cleavage, reverse transcription, and a combination thereof.

4. The method of claim 2, wherein said one or more reactions are performed in said partition.

5. The method of claim 2, wherein said one or more reactions are performed subsequent to (a).

6. The method of claim 1, further comprising using a nucleic acid barcode molecule from said plurality of nucleic acid barcode molecules and said nucleic acid molecule to generate a barcoded nucleic acid molecule.

7. The method of claim 6, further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to sequencing.

8. The method of claim 1, wherein said cell bead comprises said cell, and wherein said cell bead comprising said cell is subjected to conditions sufficient to lyse said cell to generate said one or more components.

9. The method of claim 8, wherein said cell bead is subjected to said conditions sufficient to lyse said cell in said partition.

10. The method of claim 8, wherein said conditions sufficient to lyse said cell comprise exposing said cell bead to a lysis agent.

11. The method of claim 1, wherein said partition is a droplet.

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

13. The method of claim 1, wherein one or more nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are coupled to a surface of said barcode bead or enclosed within said barcode bead.

14. The method of claim 1, wherein said partition further comprises additional reagents.

15. The method of claim 1, wherein said nucleic acid molecule is a messenger ribonucleic acid molecule.

16. The method of claim 1, further comprising recovering said nucleic acid molecule or a derivative thereof from said partition.

17. The method of claim 16, further comprising performing one or more reactions on said nucleic acid molecule or derivative thereof subsequent to recovering said nucleic acid molecule or derivative thereof from said partition.

18. The method of claim 1, wherein said barcode bead comprises a disulfide bond.

19. The method of claim 1, wherein said barcode bead is degradable upon application of a stimulus.

20. The method of claim 19, further comprising releasing said plurality of nucleic acid barcode molecules upon application of said stimulus.

21. The method of claim 19, wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.

22. The method of claim 19, wherein said stimulus is a reducing agent.

23. The method of claim 1, wherein said cell bead is degradable upon application of a stimulus.

24. The method of claim 23, wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.

25. The method of claim 23, wherein said stimulus is a reducing agent.

26. The method of claim 7, further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to one or more reactions prior to said sequencing.

27. The method of claim 26, further comprising recovering said barcoded nucleic acid molecule or derivative thereof from said partition and subsequently performing said one or more reactions.

28. The method of claim 26, wherein said one or more reactions comprises addition of a functional sequence to said barcoded nucleic acid molecule or derivative thereof, which functional sequence permits attachment of said barcoded nucleic acid molecule or derivative thereof to a flow cell of a sequencer during said sequencing.

29. The method of claim 1, wherein a given nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a identifier sequence that uniquely identifies said given nucleic acid barcode molecule among other nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules.

30. The method of claim 1, wherein said barcode bead is a gel bead.