Methods and systems for sample processing polynucleotides

• US 10,597,718 B2
• Filed: 01/11/2019
• Issued: 03/24/2020
• 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, Saxonov, Serge
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US16/246,322
• Publication Number:
  US 20190177789A1
• Time in Patent Office: 438 Days
• Field of Search: None
• 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 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 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 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse beads provides herein are degradable. For libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

Claims

1. A method for bead formation, comprising:

(a) providing a mixture comprising a plurality of bead precursor molecules and a plurality of nucleic acid molecules, wherein a nucleic acid molecule of said plurality of nucleic acid molecules comprises an adapter sequence, which adapter sequence is configured for compatibility with a nucleic acid sequencer; and

(b) in said mixture, causing linkages to form between (i) a bead precursor molecule of said plurality of bead precursor molecules and an additional bead precursor molecule of said plurality of bead precursor molecules, and (ii) said bead precursor molecule and said nucleic acid molecule of said plurality of nucleic acid molecules.

2. The method of claim 1, wherein said adapter sequence comprises at least a portion of an immobilization sequence for said nucleic acid sequencer.

3. The method of claim 1, wherein said adapter sequence comprises at least a portion of a sequencing primer binding sequence compatible with said nucleic acid sequencer.

4. The method of claim 1, wherein said plurality of bead precursor molecules comprises one or more members selected from the group consisting of monomers, polymers and crosslinkers.

5. The method of claim 1, wherein said plurality of bead precursor molecules comprises acrylamide.

6. The method of claim 1, wherein said plurality of bead precursors comprises sulfur.

7. The method of claim 1, wherein (b) is performed in a partition among a plurality of partitions.

8. The method of claim 7, wherein said partition is a droplet among a plurality of droplets.

9. The method of claim 7, further comprising, prior to (b), generating said partition with the aid of a microfluidic device.

10. The method of claim 1, wherein nucleic acid molecules of said plurality of nucleic acid molecules comprise said adapter sequence.

11. The method of claim 1, further comprising, subsequent to (b), adding a nucleic acid barcode sequence to said nucleic acid molecule or derivative thereof.

12. The method of claim 11, wherein said nucleic acid barcode sequence is from about 6 nucleotides to about 20 nucleotides in length.

13. The method of claim 11, wherein said nucleic acid barcode sequence is added to said nucleic acid molecule or derivative thereof via nucleic acid extension.

14. The method of claim 13, wherein said nucleic acid barcode sequence is added to said nucleic acid molecule or derivative thereof in a partition among a plurality of partitions, wherein said partition initially comprises a single template nucleic acid barcode molecule, and wherein said nucleic acid molecule or derivative thereof is extended when hybridized to said single template nucleic acid barcode molecule during said nucleic acid extension.

15. The method of claim 14, wherein said partition is a droplet among a plurality of droplets or a well among a plurality of wells.

16. The method of claim 11, wherein said nucleic acid barcode sequence is added to said nucleic acid molecule or derivative thereof via nucleic acid ligation.

17. The method of claim 11, further comprising adding a plurality of partial nucleic acid barcode sequences to said nucleic acid molecule or derivative thereof, wherein said nucleic acid barcode sequence comprises partial nucleic acid barcode sequences of said plurality of partial nucleic acid barcode sequences.

18. The method of claim 17, wherein partial nucleic acid barcode sequences of said plurality of partial nucleic acid barcode sequences are sequentially added to said nucleic acid molecule or derivative thereof.

19. The method of claim 17, wherein said partial nucleic acid barcode sequences of said plurality of partial nucleic acid barcode sequences comprise a different sequence.

20. The method of claim 17, wherein at least two partial barcode sequences of said plurality of partial nucleic acid barcode sequences are identical.

21. The method of claim 17, further comprising adding said plurality of partial nucleic acid barcode sequences to said nucleic acid molecule or derivative thereof in a plurality of partitions.

22. The method of claim 21, wherein said plurality of partitions is a plurality of wells.

23. The method of claim 1, further comprising adding one or more additional sequences to said nucleic acid molecule or a derivative thereof.

24. The method of claim 23, wherein said one or more additional sequences is selected from the group consisting of an immobilization sequence, a sequencing primer binding sequence, a primer sequence, a random N-mer and a poly-thymine (poly-T) sequence.

25. A method for bead formation, comprising:

(a) providing a mixture comprising a plurality of bead precursor molecules and a plurality of nucleic acid molecules, wherein a nucleic acid molecule of said plurality of nucleic acid molecules comprises a barcode sequence; and

(b) in said mixture, causing linkages to form between (i) a bead precursor molecule of said plurality of bead precursor molecules and an additional bead precursor molecule of said plurality of bead precursor molecules, and (ii) said bead precursor molecule and said nucleic acid molecule of said plurality of nucleic acid molecules.

26. The method of claim 25, wherein said plurality of bead precursor molecules comprises one or more members selected from the group consisting of monomers, polymers and crosslinkers.

27. The method of claim 25, wherein said plurality of bead precursor molecules comprises acrylamide.

28. The method of claim 25, wherein said plurality of bead precursors comprises sulfur.

29. The method of claim 25, wherein (b) is performed in a partition among a plurality of partitions.

30. The method of claim 29, wherein said partition is a droplet among a plurality of droplets.