Methods for droplet-based sample preparation

• US 9,689,024 B2
• Filed: 02/17/2015
• Issued: 06/27/2017
• 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:
  US14/624,468
• Publication Number:
  US 20150225777A1
• Time in Patent Office: 861 Days
• Field of Search: 435 61, 435 911, 435 912, 4352872, 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 sample preparation, comprising:

a) providing a droplet comprising a porous gel bead and a target nucleic acid analyte, wherein said porous gel bead comprises at least 1,000,000 oligonucleotide molecules comprising barcode sequences, wherein said oligonucleotide molecules are releasably attached to said porous gel bead, wherein said barcode sequences are the same sequence for said oligonucleotide molecules;

b) applying a stimulus to said porous gel bead to release said oligonucleotide molecules from said porous gel bead into said droplet, wherein upon release from said porous gel bead, a given oligonucleotide molecule from said oligonucleotide molecules attaches to said target nucleic acid analyte; and

c) subjecting said given oligonucleotide molecule attached to said target nucleic acid analyte to nucleic acid amplification to yield a barcoded target nucleic acid analyte.

2. The method of claim 1, wherein said droplet is a an aqueous droplet in a continuous oil phase.

3. The method of claim 1, wherein said oligonucleotide molecules are attached to the porous gel bead via a labile moiety.

4. The method of claim 3, wherein said labile moiety is a disulfide bond.

5. The method of claim 1, 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.

6. The method of claim 5, wherein said stimulus is a chemical stimulus that is a reducing agent.

7. The method of claim 6, wherein said reducing agent is dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP).

8. The method of claim 1, wherein said given oligonucleotide molecule of said oligonucleotide molecules comprises a region which functions as a primer during said nucleic acid amplification in c).

9. The method of claim 8, wherein said region which functions as said primer has a sequence for random priming.

10. The method of claim 8, wherein said primer is configured to amplify said target nucleic acid analyte, thereby producing said barcoded target nucleic acid analyte.

11. The method of claim 1, wherein said droplet further comprises a polymerase.

12. The method of claim 11, wherein said oligonucleotide molecules comprise uracil and said polymerase does not recognize uracil.

13. The method of claim 1, wherein said target nucleic acid analyte is selected from the group consisting of DNA, 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.

14. The method of claim 1, wherein said oligonucleotide molecules are coupled to said porous gel bead via a covalent bond.

15. The method of claim 1, wherein said oligonucleotide molecules are reversibly immobilized to said porous gel bead.

16. The method of claim 1, wherein said droplet in (a) comprises a plurality of target nucleic acid analytes, which plurality of target nucleic acid analytes comprises said target nucleic acid analyte.

17. The method of claim 16, wherein each of said plurality of target nucleic acid analytes attaches to an individual oligonucleotide molecule of said plurality of oligonucleotide molecules.

18. The method of claim 16, further comprising fragmenting a nucleic acid sample to yield said plurality of target nucleic acid analytes.

19. The method of claim 1, wherein said given oligonucleotide molecule from said oligonucleotide molecules attaches to said target nucleic acid analyte by hybridization.

20. The method of claim 1, further comprising, prior to (a), providing a nucleic acid sample and fragmenting said nucleic acid sample to yield said target nucleic acid analyte.

21. The method of claim 1, wherein said porous gel bead comprises a polymer gel.

22. The method of claim 21, wherein said polymer gel is a polyacrylamide.