Methods for droplet-based sample preparation

• US 9,695,468 B2
• Filed: 02/17/2015
• Issued: 07/04/2017
• Est. Priority Date: 08/14/2012
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Schnall-Levin, Michael
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/624,473
• Publication Number:
  US 20150225778A1
• Time in Patent Office: 868 Days
• Field of Search: 435 61, 435 911, 435 912, 4352872, 4352885, 536 243, 536 2433, 422 681, 506 16, 506 30, 506 31
• 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: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/508 rigid containers not provided for above
• CPC Class Codes: B01L 3/523 with means for closing or opening
• 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 microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.

Claims

1. A method for droplet generation, comprising:

(a) providing at least 1,000,000 oligonucleotide molecules comprising barcode sequences, wherein said barcode sequences are the same sequence for said at least 1,000,000 oligonucleotide molecules, wherein said at least 1,000,000 oligonucleotide molecules are releasably attached to a bead, wherein said bead is porous;

(b) combining said at least 1,000,000 oligonucleotide molecules and a sample comprising a nucleic acid analyte each in an aqueous phase at a first junction of two or more channels of a microfluidic device to form an aqueous mixture comprising said at least 1,000,000 oligonucleotide molecules attached to said bead and said sample; and

(c) generating a droplet comprising said at least 1,000,000oligonucleotide molecules attached to said bead and said sample comprising said nucleic acid analyte by contacting said aqueous mixture with an immiscible continuous phase at a second junction of two or more channels of said microfluidic device.

2. The method of claim 1, further comprising, in (b), combining said at least 1,000,000 oligonucleotide molecules attached to said bead, said nucleic acid analyte and one or more reagents necessary for amplification of said nucleic acid analyte at said first junction to form said aqueous mixture comprising said at least 1,000,000 oligonucleotide molecules attached to said bead, said nucleic acid analyte and said one or more reagents.

3. The method of claim 2, wherein in (c), said droplet further comprises said one or more reagents.

4. The method of claim 3, wherein said one or more reagents comprises a polymerase.

5. The method of claim 4, wherein said polymerase is unable to recognize uracil.

6. The method of claim 1, wherein said bead comprises a polyacrylamide.

7. The method of claim 1, wherein said bead is a gel bead.

8. The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules comprise uracil.

9. The method of claim 1, wherein a given oligonucleotide molecule of said at least 1,000,000 oligonucleotide molecules comprises a region which functions as a primer.

10. The method of claim 9, wherein said region which functions as said primer has a sequence for random priming.

11. The method of claim 9, further comprising, after (c), amplifying said nucleic acid analyte with said primer.

12. The method of claim 1, wherein said nucleic acid analyte is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), amplicons, synthetic polynucleotides, polynucleotides, oligonucleotides, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.

13. The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules are attached to said bead via a chemical cross-linker.

14. The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules are attached to said bead via a disulfide bond.

15. The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules are attached to said bead via a covalent bond.

16. The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules are attached to said bead via a labile moiety.

17. The method of claim 1, wherein said bead is degradable upon application of a stimulus.

18. The method of claim 17, further comprising applying said stimulus to the droplet to release said at least 1,000,000 oligonucleotide molecules from said bead into said droplet.

19. The method of claim 18, wherein said stimulus is selected from the group consisting of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, and a photo stimulus.

20. The method of claim 19, wherein said stimulus is a chemical stimulus that is a reducing agent.

21. The method of claim 1, wherein subsequent to generating said droplet in (c), a given oligonucleotide molecule of said at least 1,000,000 oligonucleotide molecules attaches to said nucleic acid analyte, and wherein said given oligonucleotide molecule attached to said given nucleic acid analyte is subjected to nucleic acid amplification to yield a barcoded nucleic acid analyte.

22. The method of claim 1, wherein said bead comprises a chemical cross-linker.

23. The method of claim 22, wherein said chemical cross-linker is a disulfide bond.