COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20140378349A1
• 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, Jarosz, Mirna, Masquelier, Donald, Saxonov, Serge, Merrill, Landon, Price, Andrew, Hardenbol, Paul, Li, Yuan
• Application Number:
  US14/316,416
• Publication Number:
  US 20140378349A1
• 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 2219/00592 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 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: C12Q 2600/16 Primer sets for multiplex assays

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 generating functionalized beads comprising:

a. mixing a plurality of polymers or monomers with one or more oligonucleotides;

b. crosslinking the plurality of polymers or monomers such that disulfide bonds form between the plurality of polymers or monomers, thereby forming hardened beads; and

c. causing covalent linkages to form between the one or more oligonucleotides and the plurality of polymers or monomers.

2. The method of claim 1, wherein the plurality of polymers or monomers comprise acrylamide.

3. The method of claim 1, wherein steps b and c are performed contemporaneously.

4. The method of claim 1, wherein steps b and c are performed sequentially.

5. The method of claim 1, wherein the one or more oligonucleotides comprise a primer.

6. The method of claim 5, wherein the primer is linked to an acrydite moiety.

7. The method of claim 5, wherein the primer is a universal primer.

8. The method of claim 5, wherein the primer is a sequencing primer.

9. The method of claim 1, further comprising:

attaching one or more first additional oligonucleotides to the one or more oligonucleotides.

10. The method of claim 9, wherein the one or more first additional oligonucleotides is a barcode sequence, thereby forming a barcoded bead.

11. The method of claim 10, wherein the barcode sequence is between about 6 nucleotides and about 20 nucleotides in length.

12. The method of claim 5, further comprising creating a mixture by combining the functionalized beads with a plurality of first additional oligonucleotides.

13. The method of claim 12, further comprising partitioning the mixture into a plurality of partitions such that, on average, each partition comprises no more than one of the plurality of first additional oligonucleotides.

14. The method of claim 13, further comprising amplifying the plurality of first additional oligonucleotides within the partitions, thereby producing beads comprising amplified first oligonucleotides.

15. The method of claim 13, wherein the partitions are aqueous droplets within a water-in-oil emulsion.

16. The method of claim 13, wherein the partitions are generated by a microfluidic device.

17. The method of claim 13, wherein the partitions are generated by a bulk emulsification process.

18. The method of claim 14, further comprising pooling the contents of the partitions into a common vessel.

19. The method of claim 18, further comprising separating the beads comprising amplified first oligonucleotides from the contents of the partitions.

20. The method of claim 14, further comprising using a capture primer during the amplifying, wherein the capture primer is attached to a capture moiety.

21. The method of claim 14, further comprising hybridizing a probe to the amplified first oligonucleotides wherein the probe is attached to a capture moiety.

22. The method of claim 20 or 21, wherein the capture moiety is biotin, streptavidin, or glutathioine-S-transferase (GST).

23. The method of claim 18, further comprising attaching one or more second additional oligonucleotides to the amplified first oligonucleotides.

24. The method of claim 23, wherein the one or more second additional oligonucleotides comprise a random and/or a pseudo random N-mer sequence.

25. The method of claim 23, wherein the one or more second additional oligonucleotides comprise a primer binding site.

26. The method of claim 25, wherein the primer binding site further comprises a universal sequence portion.

27. The method of claim 26, wherein the universal sequence portion is compatible with a sequencing device.

28. The method of claim 26, wherein the universal sequence portion further comprises a subsection of uracil containing nucleotides.

29. The method of claim 25, wherein the primer binding site further comprises uracil containing nucleotides.