COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20180051321A1
• Filed: 09/28/2017
• Published: 02/22/2018
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Masquelier, Donald, Saxonov, Serge, Merrill, Landon, Price, Andrew, Hardenbol, Paul, Li, Yuan
• Application Number:
  US15/718,893
• Publication Number:
  US 20180051321A1
• Time in Patent Office: Days
• 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 for generating a plurality of beads for use in nucleic acid barcoding, comprising:

using combinatorial synthesis to generate a plurality of nucleic acid barcode molecules coupled to a plurality of beads, wherein said plurality of nucleic acid barcode molecules comprises a plurality of barcode sequences that are different across said plurality of beads,

wherein a given bead of said plurality of beads comprises nucleic acid barcode molecules coupled thereto, wherein a given nucleic acid barcode molecule of said nucleic acid barcode molecules comprises a barcode sequence comprising a first nucleic acid barcode segment and a second nucleic acid barcode segment,

wherein said first nucleic acid barcode segment is the same across said nucleic acid barcode molecules coupled to said given bead.

2. The method of claim 1, wherein said combinatorial synthesis comprises coupling a first nucleic acid molecule comprising said first nucleic acid barcode segment to said given bead, and coupling a second nucleic acid molecule comprising said second nucleic acid barcode segment to said first nucleic acid molecule.

3. The method of claim 2, wherein said second nucleic acid molecule is coupled to said first nucleic acid molecule through ligation.

4. The method of claim 2, wherein said combinatorial synthesis comprises providing said given bead in a first partition among a first plurality of partitions, wherein said first partition comprises said first nucleic acid molecule, and coupling said first nucleic acid molecule to said given bead in said first partition.

5. The method of claim 4, wherein said first plurality of partitions is a plurality of wells.

6. The method of claim 4, further comprising directing said given bead to a second partition among a second plurality of partitions, wherein said second partition comprises said second nucleic acid molecule, and coupling said second nucleic acid molecule to said first nucleic acid molecule.

7. The method of claim 6, wherein said second plurality of partitions is a plurality of wells.

8. The method of claim 1, wherein at least a subset of said nucleic acid barcode molecules is in an interior of said given bead.

9. The method of claim 8, wherein said at least said subset of said nucleic acid barcode molecules is coupled to said interior of said given bead.

10. The method of claim 9, wherein said at least said subset of said nucleic acid barcode molecules is reversibly coupled to said interior of said given bead.

11. The method of claim 10, wherein said at least said subset of said nucleic acid barcode molecules is reversibly coupled to said interior of said given bead through chemical cross-linkers.

12. The method of claim 9, wherein (i) a first subset said nucleic acid barcode molecules is coupled to said interior of said given bead and (ii) a second subset of said nucleic acid barcode molecules is coupled to an exterior of said given bead.

13. The method of claim 1, wherein said given nucleic acid barcode molecule comprises a targeted primer sequence or a variable primer sequence.

14. The method of claim 1, wherein said nucleic acid barcode molecules comprise unique identifiers that are different across said nucleic acid barcode molecules coupled to said given bead.

15. The method of claim 1, wherein said nucleic acid barcode molecules comprises at least 100,000 nucleic acid barcode molecules.

17. The method of claim 1, wherein said plurality of beads comprises at least 100,000 nucleic acid barcode molecules with different barcode sequences.

18. The method of claim 17, wherein said plurality of beads comprises at least 1,000,000 nucleic acid barcode molecules with different barcode sequences.

19. The method of claim 1, wherein said plurality of beads is a plurality of gel beads.

20. The method of claim 19, wherein each of said plurality of gel beads is dissolvable or disruptable.

21. The method of claim 19, wherein said plurality of gel beads is formed upon polymerization of molecular precursors within a plurality of droplets comprising said plurality of nucleic acid barcode molecules.

22. The method of claim 1, wherein said plurality of beads is uniform in size.

23. The method of claim 1, wherein said plurality of beads has a coefficient of variation in cross-sectional dimension of less than 30%.

24. The method of claim 1, wherein said given bead has a diameter less than 100 micrometers.

25. The method of claim 24, wherein said diameter is less than 60 micrometers.

26. The method of claim 1, wherein said nucleic acid barcode molecules is releasable from said given bead.

27. The method of claim 1, wherein said first nucleic acid barcode segment or said second nucleic acid barcode segment of said given bead is different from a corresponding segment of another bead of said plurality of beads.

28. The method of claim 1, wherein said first nucleic acid barcode segment or said second nucleic acid barcode segment is from 4 to 20 nucleic acid bases in length.

29. The method of claim 1, wherein said barcode sequence is at least 15 nucleic acid bases in length.

31. The method of claim 1, wherein said first nucleic acid barcode segment is separated from said second nucleic acid barcode segment by a nucleic acid sequence that is the same across at least a subset of said plurality of beads.

32. The method of claim 31, wherein said nucleic acid sequence is at least 2 nucleic acid bases in length.

16. (canceled)

30. (canceled)