COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20150005199A1
• Filed: 06/26/2014
• Published: 01/01/2015
• 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:
  US14/316,398
• Publication Number:
  US 20150005199A1
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 2219/00722 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
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

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 bead that is covalently linked to a plurality of oligonucleotides comprising an identical barcode sequence and a variable domain.

2. The composition of claim 1, wherein the plurality of oligonucleotides further comprises a primer binding site.

3. The composition of claim 1, wherein the plurality of oligonucleotides further comprises a universal primer.

4. The composition of claim 1, wherein the identical barcode sequence is between about 6 nucleotides and about 20 nucleotides in length.

5. The composition of claim 1, wherein the plurality of oligonucleotides are covalently linked to the bead by disulfide linkages.

6. The composition of claim 1, wherein the bead further comprises a cystamine or a modified cystamine.

7. The composition of claim 1, wherein the bead is capable of being substantially dissolved by a reducing agent.

8. The composition of claim 1, wherein the bead comprises at least about 1,000,000 oligonucleotides comprising an identical barcode sequence.

9. The composition of claim 1, wherein at least about 30% of the plurality of oligonucleotides comprise variable domains with different sequences.

10. The composition of claim 1, wherein the variable domain is a random Nmer.

11. The composition of claim 1, wherein the bead is covalently linked to the plurality of oligonucleotides through a cleavable linkage.

12. The composition of claim 11, wherein the cleavable linkage is selected from a chemically cleavable linkage, a photocleavable linkage and a thermally cleavable linkage.

13. A composition, comprising a bead, wherein:

the bead comprises a plurality of more than 1,000,000 oligonucleotides, wherein each of the plurality of oligonucleotides comprises a constant region and a variable region; and

the bead is capable of being substantially dissolved with a reducing agent.

14. The composition of claim 13, wherein each oligonucleotide has an identical constant region.

15. The composition of claim 13, wherein at least 25% of the more than 1,000,000 oligonucleotides have an identical constant region.

16. The composition of claim 13, wherein the constant region comprises a barcode sequence.

17. The composition of claim 13, wherein at least 25% of the more than 1,000,000 oligonucleotides have a variable region comprising a different sequence.

21. A library comprising at least about 1,000,000 of the beads of claim 13, wherein at least about 25% of the beads comprise oligonucleotides with different nucleotide sequences.

18. A composition, comprising:

a plurality of beads;

wherein each of the plurality of beads comprises a plurality of oligonucleotides releasably coupled thereto;

wherein the plurality of oligonucleotides associated with an individual bead from the plurality of beads comprises a common barcode domain and a variable domain; and

wherein the common barcode domain is different between two or more beads in the plurality of beads.

19. The composition of claim 18, wherein the plurality of beads comprises at least about 10,000 different barcode domains coupled to different beads in the plurality of beads.

20. The composition of claim 18, wherein each of the plurality of beads comprises at least about 1,000,000 oligonucleotides releasably coupled thereto.