Methods and systems for processing polynucleotides

• US 10,626,458 B2
• Filed: 06/07/2019
• Issued: 04/21/2020
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Christopher, Schnall-Levin, Michael, Price, Andrew, Hardenbol, Paul, Li, Yuan
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/435,362
• Publication Number:
  US 20190316197A1
• Time in Patent Office: 319 Days
• 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 libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For 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 composition comprising:

a plurality of beads comprising a plurality of oligonucleotide molecules covalently attached thereto;

wherein said plurality of oligonucleotide molecules comprises a plurality of barcode sequences;

wherein said plurality of beads comprises N×

M beads;

wherein said plurality of barcode sequences comprises N×

M different barcode sequences;

wherein each barcode sequence of said plurality of barcode sequences comprises a first barcode sequence segment (B1) and a second barcode sequence segment (B2);

wherein each oligonucleotide molecule of said plurality of oligonucleotide molecules comprises a sequence having the structure A-B1-L-B2-C, wherein (i) A is an adapter sequence, (ii) L is a linking sequence, and (iii) C is a capture sequence selected from the group consisting of a poly-T sequence, a random N-mer sequence, and a gene-specific primer sequence;

wherein said plurality of barcode sequences comprises;

(i) M different first barcode sequence segments at least m nucleotides in length, and (ii) N different second barcode sequence segments at least n nucleotides in length; and

wherein M=96, N=96, m≥

6, and n≥

6.

2. The composition of claim 1, wherein M=384 and N=384.

3. The composition of claim 1, wherein said linking sequence is 1-10 nucleotides in length.

4. The composition of claim 1, wherein said linking sequence is 2-6 nucleotides in length.

5. The composition of claim 1, wherein said linking sequence is 10 or more nucleotides in length.

6. The composition of claim 1, wherein said adapter sequence is identical across said plurality of oligonucleotide molecules.

7. The composition of claim 6, wherein said adapter sequence comprises one or more functional sequences.

8. The composition of claim 7, wherein said one or more functional sequences comprise a primer sequence or a primer binding sequence.

9. The composition of claim 8, wherein said primer sequence or said primer binding sequence is a sequencing primer sequence or a sequencing primer binding sequence.

10. The composition of claim 9, wherein said sequencing primer sequence or said sequencing primer binding sequence is a R1 sequence, a R2 sequence, a partial R1 sequence, or a partial R2 sequence.

11. The composition of claim 7, wherein said one or more functional sequences comprise a sequence configured to attach to a flow cell of a sequencer.

12. The composition of claim 11, wherein said sequence configured to attach to the flow cell of a sequencer is a P5 sequence, a P7 sequence, a partial P5 sequence, or a partial P7 sequence.

13. The composition of claim 1, wherein said plurality of oligonucleotide molecules are single stranded.

14. The composition of claim 1, wherein said plurality of beads are a plurality of gel beads.

15. The composition of claim 14, wherein said plurality of gel beads are degradable upon application of a stimulus.

16. The composition of claim 15, wherein said plurality of gel beads comprise a plurality of crosslinks that are degradable upon application of a stimulus.

17. The composition of claim 15, wherein said stimulus is a thermal stimulus, a chemical stimulus, a biological stimulus, or a photo stimulus.

18. The composition of claim 16, wherein said plurality of crosslinks comprise a disulfide bond and wherein said stimulus is a reducing agent.

19. The composition of claim 14, wherein said plurality of oligonucleotides are releasable from said gel bead upon application of a stimulus.

20. The composition of claim 19, wherein said plurality of oligonucleotide molecules are attached to said gel bead via a disulfide bond and wherein said stimulus is a reducing agent.

21. The composition of claim 1, wherein said plurality of oligonucleotides are releasable from said bead upon application of a stimulus.

22. The composition of claim 21, wherein said stimulus is a thermal stimulus, a chemical stimulus, a biological stimulus, or a photo-stimulus.

23. The composition of claim 22, wherein said plurality of oligonucleotide molecules are attached to said bead via a disulfide bond and wherein said stimulus is a reducing agent.

24. The composition of claim 1, wherein said plurality of beads comprises M×

N×

Z beads;

wherein said plurality of barcode sequences comprises M×

N×

Z different barcode sequences;

wherein each barcode sequence of said plurality of barcode sequences further comprises a third barcode sequence segment (B3);

wherein each oligonucleotide molecule of said plurality of oligonucleotide molecules comprises a sequence having the structure A-B1-L1-B2-L2-B3-C, wherein (i) L1 is a first linking sequence, and (ii) L2 is a second linking sequence;

wherein said plurality of barcode sequences further comprises;

(i) Z different third barcode sequence segments at least z nucleotides in length; and

wherein Z=96 and z≥

6.

25. The composition of claim 24, wherein L1 and L2 are different sequences.

26. The composition of claim 24, wherein L1 and L2 are each 1-10 nucleotides in length.

27. The composition of claim 24, wherein L1 and L2 are each 10 or more nucleotides in length.

28. The composition of claim 1, wherein said plurality of oligonucleotide molecules comprise a uracil containing nucleotide.

29. The composition of claim 1, wherein each bead of said plurality of beads comprises 1,000,000 oligonucleotide molecules covalently attached thereto.

30. A composition comprising:

a plurality of beads comprising a plurality of oligonucleotide molecules covalently attached thereto;

wherein said plurality of oligonucleotide molecules comprises a plurality of barcode sequences;

wherein said plurality of beads comprises N×

M beads;

wherein said plurality of barcode sequences comprises N×

M different barcode sequences;

wherein each barcode sequence of said plurality of barcode sequences comprises a first barcode sequence segment (B1) and a second barcode sequence segment (B2);

wherein each oligonucleotide molecule of said plurality of oligonucleotide molecules comprises a sequence having the structure A-B1-L-B2-T, wherein (i) A is an adapter sequence, (ii) L is a linking sequence, and (iii) T is a poly-T sequence;

wherein said plurality of barcode sequences comprises;

(i) M different first barcode sequence segments at least m nucleotides in length, and (ii) N different second barcode sequence segments at least n nucleotides in length; and

wherein M=96, N=96, m≥

6, and n≥

6.