COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20140378350A1
• Filed: 06/26/2014
• Published: 12/25/2014
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Masquelier, Donald, Saxonov, Serge, Merrill, Landon, Price, Andrew, Hardenbol, Paul, Li, Yuan
• Granted Patent: US 10,221,442 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 2219/00722 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 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

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 amplifying a template nucleic acid sequence, comprising:

(a) co-partitioning a template nucleic acid sequence and a bead comprising a plurality of oligonucleotides releasably attached thereto into a partition, the plurality of oligonucleotides comprising a primer sequence complementary to one or more regions of the template nucleic acid sequence and a common sequence;

(b) annealing the primer sequence to the template nucleic acid sequence; and

(c) extending the primer sequence to produce one or more first copies of at least a portion of the template nucleic acid sequence, the one or more first copies comprising the primer sequence and the common sequence.

2. The method of claim 1, wherein the primer sequence comprises a variable primer sequence.

3. The method of claim 1, wherein the primer sequence comprises a targeted primer sequence.

4. The method of claim 1, wherein the partition comprises a droplet in an emulsion.

5. The method of claim 1, wherein prior to the annealing step, the plurality of oligonucleotides are released from the bead into the partition.

6. The method of claim 1, comprising providing a polymerase enzyme in the partition.

7. The method of claim 1, wherein the extending step comprises extending the primer sequence using a strand displacing polymerase enzyme.

8. The method of claim 7, wherein the strand displacing polymerase enzyme comprises a thermostable strand displacing polymerase enzyme.

9. The method of claim 6, wherein the polymerase enzyme comprises an exonuclease deficient polymerase enzyme.

10. The method of claim 1, wherein the oligonucleotides are exonuclease resistant.

11. The method of claim 10, wherein the oligonucleotides comprise one or more phosphorothioate linkages.

12. The method of claim 11, wherein the one or more phosphorothioate linkages comprise a phosphorothioate linkage at a terminal internucleotide linkage in the oligonucleotides.

13. The method of claim 8, wherein the polymerase enzyme has substantially no exonuclease activity.

14. The method of claim 2, wherein the variable primer sequence comprises a random N-mer.

15. The method of claim 1, further comprising the steps of:

(d) annealing one or more variable primer sequences to the one or more first copies; and

(e) extending the one or more variable primer sequences to produce one or more second copies from the one or more first copies, the one or more second copies comprising the one or more variable primer sequences and the common sequence.

16. The method of claim 15, wherein the one or more second copies further comprise a sequence complementary to at least a portion of an individual first copy of the one or more first copies, and a sequence complementary to an individual variable sequence of the one or more variable primer sequences.

17. The method of claim 15, comprising repeating steps (a)-(e) to produce amplified nucleic acids.

18. The method of claim 15, wherein the plurality of oligonucleotides comprises a sequence segment that is not copied during the extending step (e).

19. The method of claim 18, wherein the sequence segment that is not copied comprises one or more uracil containing nucleotides.

20. The method of claim 15, wherein the one or more second copies preferentially form a hairpin molecule under annealing conditions.

21. A method of amplifying a plurality of different nucleic acids, comprising:

partitioning different nucleic acids into separate first partitions, each first partition comprising a second partition having a plurality of oligonucleotides releasably associated therewith, the plurality of oligonucleotides associated with a given second partition comprising a variable primer sequence and a barcode sequence, and the plurality of oligonucleotides associated with different second partitions comprising different barcode sequences;

releasing oligonucleotides associated with the plurality of second partitions into the first partitions;

annealing variable primer sequences within the first partitions to nucleic acids within the first partitions; and

extending the variable primer sequences to produce one or more copies of at least a portion of the nucleic acids within the first partitions, the one or more copies comprising the oligonucleotides released into the first partitions.

22. The method of claim 21, wherein the first partitions comprise droplets in an emulsion, and the plurality of second partitions comprise beads.

23. The method of claim 22, wherein each bead comprises more than 100,000 oligonucleotides associated therewith.

24. The method of claim 22, wherein each bead comprises more than 1,000,000 oligonucleotides associated therewith.

25. The method of claim 21, wherein the plurality of second partitions comprise at least 1000 different barcode sequences.

26. The method of claim 21, wherein the plurality of second partitions comprise at least 10,000 different barcode sequences.

27. The method of claim 21, wherein the plurality of second partitions comprise at least 100,000 different barcode sequences.

28. A method of whole genome amplification comprising:

(a) hybridizing a random primer to a genomic nucleic acid, wherein the random primer is attached to a universal nucleic acid sequence and a nucleic acid barcode sequence, wherein the universal nucleic acid sequence comprises one or more uracil containing nucleotides;

(b) extending the random primer to form an amplified product; and

(c) causing the amplified product to undergo an intramolecular hybridization reaction, thereby forming a partial hairpin molecule.

29. The method of claim 28, wherein the random primer is a random N-mer sequence.

30. The method of claim 28, wherein the universal nucleic acid sequence comprises a segment of at least 10 nucleotides that do not comprise uracil.

31. The method of claim 28, further comprising performing the method in the presence of an oligonucleotide blocker.

32. The method of claim 31, wherein the oligonucleotide blocker comprises a C3 spacer (/3SpC3/), a Dideoxy-C (/3ddC/), or a 3′

phosphate.

33. The method of claim 31, wherein the oligonucleotide blocker is capable of hybridizing to at least a portion of the universal nucleic acid sequence.

34. A method of amplifying nucleic acids, comprising:

fragmenting a genomic component into a plurality of first fragments;

co-partitioning the first fragments into a plurality of partitions with a plurality of oligonucleotides, the plurality of oligonucleotides within each of the plurality of partitions comprising a primer sequence and a common sequence;

annealing primer sequences in each partition of the plurality of partitions to a plurality of different regions of the first fragments within each partition of the plurality of partitions; and

extending the primer sequences along the plurality of first fragments to produce amplified first fragments within each partition of the plurality of partitions.

35. The method of claim 34, wherein the amplified first fragments within the plurality of partitions comprise at least 1×

coverage of the genomic component.

36. The method of claim 34, wherein the amplified first fragments within the plurality of partitions comprise at least 2×

coverage of the genomic component.

37. The method of claim 34, wherein the amplified first fragments within the plurality of partitions comprise at least 10×

coverage of the genomic component.

38. The method of claim 34, wherein the genomic component comprises a chromosome.

39. The method of claim 34, wherein the genomic component comprises a whole genome of an organism.