COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20140378322A1
• Filed: 06/26/2014
• Published: 12/25/2014
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Masquelier, Donald, Saxonov, Serge, Hardenbol, Paul, Jarosz, Mirna, Merrill, Landon, Price, Andrew, Li, Yuan
• Application Number:
  US14/316,463
• Publication Number:
  US 20140378322A1
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• 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: 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 2535/122 Massive parallel sequencing
• 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: C40B 50/16 involving encoding steps

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 method of characterizing a nucleic acid segment, comprising:

co-partitioning the nucleic acid segment and a bead comprising a plurality of oligonucleotides that comprise a common nucleic acid barcode sequence into a partition;

attaching the oligonucleotides to fragments of the nucleic acid segment or to copies of portions of the nucleic acid segment, thereby attaching the common nucleic acid barcode sequence to the fragments of the nucleic acid segment or to the copies of the portions of the nucleic acid segment;

sequencing the fragments of the nucleic acid segment or the copies of the portions of the nucleic acid segment and the attached common nucleic acid barcode sequence; and

characterizing the fragments or the copies of the portions of the nucleic acid segment as being linked within the nucleic acid segment based at least in part, upon their attachment to the common nucleic acid barcode sequence.

2. The method of claim 1, wherein the nucleic acid segment and the bead are co-partitioned into a droplet in an emulsion.

3. The method of claim 1, wherein the nucleic acid segment and the bead are co-partitioned into a microcapsule.

4. The method of claim 1, wherein the fragments of the nucleic acid segment comprise overlapping fragments of the nucleic acid segment.

5. The method of claim 1, wherein the fragments of the nucleic acid segment comprise greater than 2×

coverage of the nucleic acid segment.

6. The method of claim 1, wherein the fragments of the nucleic acid segment comprise greater than 10×

coverage of the nucleic acid segment.

7. The method of claim 1, wherein the plurality of oligonucleotides is releasably attached to the bead.

8. The method of claim 7, wherein the plurality of oligonucleotides is releasable from the bead upon application of a stimulus to the bead.

9. The method of claim 8, wherein the stimulus is selected from a thermal stimulus, a photo stimulus and a chemical stimulus.

10. The method of claim 8, wherein the application of the stimulus results in one or more of cleavage of a linkage between the oligonucleotides and the bead, and degradation of the bead to release the oligonucleotides from the bead.

11. The method of claim 1, wherein the bead comprises at least about 10,000 oligonucleotides attached thereto.

12. The method of claim 1, wherein the bead comprises at least about 100,000 oligonucleotides attached thereto.

13. The method of claim 1, wherein the bead comprises at least about 1,000,000 oligonucleotides attached thereto.

14. The method of claim 1, wherein the bead comprises at least about 10,000,000 oligonucleotides attached thereto.

15. The method of claim 1, wherein the bead comprises at least about 100,000,000 oligonucleotides attached thereto.

16. The method of claim 1, wherein the plurality of oligonucleotides further comprise one or more functional sequences.

17. The method of claim 16, wherein the one or more functional sequences are selected from primer sequences, primer annealing sequences, and immobilization sequences.

18. The method of claim 1, wherein the sequencing step comprises a sequencing by synthesis process.

19. The method of claim 1, wherein:

the plurality of oligonucleotides comprises a primer sequence capable of annealing with a portion of the nucleic acid segment or a complement thereof; and

the attaching step comprises extending the primer sequence to replicate at least a portion of the nucleic acid segment or complement thereof, to produce a copy of a portion of the nucleic acid segment or complement thereof comprising the common nucleic acid barcode sequence.

20. The method of claim 19, wherein the plurality of oligonucleotides further comprises at least a first sequencing primer sequence.

21. The method of claim 1, comprising:

co-partitioning a plurality of nucleic acid segments with a plurality of different beads into a plurality of separate partitions, such that each partition of a plurality of different partitions of the plurality of separate partitions contains a single bead, wherein each bead comprises a plurality of oligonucleotides attached thereto, the plurality of oligonucleotides on each bead comprising a common barcode sequence, and the plurality of different beads comprises a plurality of different barcode sequences;

attaching barcode sequences in each partition to fragments of the plurality of nucleic acid segments or to copies of portions of the plurality of nucleic acid segments within the plurality of separate partitions;

pooling the fragments of the plurality of nucleic acid segments or the copies of the portions of the plurality of nucleic acid segments from the plurality of separate partitions;

sequencing the fragments of the plurality of nucleic acid segments or the copies of the portions of the plurality of nucleic acid segments and any associated barcode sequences to provide sequenced fragments or sequenced copies; and

characterizing the sequenced fragments or sequenced copies as deriving from a common nucleic acid segment, based in part upon such sequenced fragments or such sequenced copies comprising a common barcode sequence.

22. The method of claim 21, wherein the plurality of nucleic acid segments comprise fragments of at least a portion of a genome, and further comprising the step of assembling sequences from the sequenced fragments or sequenced copies to provide a contiguous sequence of the at least a portion of the genome.

23. The method of claim 21, wherein the characterizing step comprises characterizing the fragments of the plurality of nucleic acid segments or the copies of the portions of the nucleic acid segments based in part upon each of a nucleotide sequence of the fragments of the plurality of nucleic acid segments or the copies of the portions of the plurality of nucleic acid segments, and the sequenced fragments or sequenced copies comprising a common barcode sequence.

24. The method of claim 22, wherein the assembling step comprises assembling sequences from the sequenced fragments or sequenced copies based in part upon each of a nucleotide sequence of the sequenced fragments or sequenced copies, and the sequenced fragments or sequenced copies comprising a common barcode sequence.

25. The method of claim 21, wherein the plurality of different beads comprises at least 1,000 different barcode sequences.

26. The method of claim 21, wherein the plurality of different beads comprises at least 10,000 different barcode sequences.

27. The method of claim 21, wherein the plurality of different beads comprises at least 100,000 different barcode sequences.

28. The method of claim 21, wherein the plurality of different beads comprises at least 1,000,000 different barcode sequences.

29. The method of claim 21, wherein two or more partitions of the plurality of separate partitions comprise beads that comprise the same barcode sequence.

30. The method of claim 21, wherein at least 1% of the plurality of separate partitions comprises beads having the same barcode sequence.

31. A method of characterizing a target nucleic acid, comprising:

partitioning first fragments of the target nucleic acid into a plurality of droplets, each droplet of the plurality of droplets comprising a bead having a plurality of oligonucleotides attached thereto, the oligonucleotides attached to a given bead comprising a common barcode sequence;

attaching the common barcode sequence to second fragments of the first fragments;

pooling the plurality of droplets;

sequencing the second fragments and attached barcode sequences; and

mapping a plurality of the second fragments to one or more of the first fragments based, at least in part, upon the plurality of second fragments comprising a common barcode sequence.

32. A method of sequencing nucleic acids, comprising:

providing a plurality of target nucleic acid sequences;

separating the target nucleic acid sequences into a plurality of separate partitions, each partition of the plurality of separate partitions comprising one or more target nucleic acid sequences, and a bead having a plurality of oligonucleotides attached thereto, the oligonucleotides attached to a given bead comprising a common barcode sequence;

attaching the oligonucleotides to fragments of the one or more target nucleic acid sequences or to copies of portions of the one or more target nucleic acid sequences within a partition, thereby attaching the common barcode sequence to the fragments of the one or more target nucleic acid sequences or to the copies of the portions of the one or more target nucleic acid sequences;

pooling the plurality of separate partitions; and

sequencing the fragments of the one or more target nucleic acid sequences or the copies of the portions of the one or more target nucleic acid sequences and attached barcode sequences to provide barcoded fragment sequences or barcoded copy sequences.

33. The method of claim 32, further comprising assembling the barcoded fragment sequences or barcoded copy sequences into one or more contiguous nucleic acid sequences based, in part, upon a barcode portion of the barcoded fragment sequences or barcoded copy sequences.

34. A method of characterizing a nucleic acid segment, comprising:

co-partitioning the nucleic acid segment, and a bead comprising a plurality of oligonucleotides that comprise a common nucleic acid barcode sequence, into a first droplet;

attaching the oligonucleotides to fragments of the nucleic acid segment or to copies of portions of the nucleic acid segment, thereby attaching the common nucleic acid barcode sequence to the fragments of the nucleic acid segment or to the copies of the portions of the nucleic acid segment;

sequencing the fragments of the nucleic acid segment or the copies of the portions of the nucleic acid segment and attached common nucleic acid barcode sequence to provide a plurality of barcoded fragment sequences or a plurality of barcoded copy sequences; and

assembling the plurality of the barcoded fragment sequences or the plurality barcoded copy sequences into one or more contiguous nucleic acid sequences based at least in part on the common nucleic acid barcode sequence.

35. The method of claim 34, wherein the assembling step comprises assembling the plurality of the barcoded fragment sequences or the plurality of barcoded copy sequences based in part upon a nucleic acid sequence of non-barcode portion of the barcoded fragment sequences or the barcoded copy sequences.

36. A method of sequencing nucleic acids, comprising:

providing a plurality of target nucleic acid sequences;

separating the target nucleic acid sequences into a plurality of separate partitions, each partition of the plurality of separate partitions comprising one or more target nucleic acid sequences, and a plurality of oligonucleotides, the oligonucleotides in a given partition comprising a common barcode sequence, and wherein the plurality of separate partitions comprise at least 10,000 different barcode sequences;

attaching the common barcode sequence in each partition to fragments of the one or more target nucleic acid sequences or to copies of portions of the one or more target nucleic acid sequences within the partition;

pooling the plurality of separate partitions; and

sequencing the fragments of the one or more target nucleic acid sequences or the copies of the portions of the one or more target nucleic acid sequences and attached barcode sequences.

37. The method of claim 36, wherein the plurality of separate partitions comprises at least 100,000 different barcode sequences.