CAPSULE ARRAY DEVICES AND METHODS OF USE

• US 20170356027A1
• Filed: 08/25/2017
• Published: 12/14/2017
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Saxonov, Serge, Schnall-Levin, Michael
• Application Number:
  US15/687,357
• Publication Number:
  US 20170356027A1
• Time in Patent Office: Days
• 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 processing a nucleic acid sample, comprising:

(a) providing at least 1,000 oligonucleotide molecules coupled to a bead, wherein the at least 1,000 oligonucleotide molecules comprise barcode sequences, which barcode sequences are the same sequence for the at least 1,000 oligonucleotide molecules;

(b) combining the at least 1,000 oligonucleotide molecules and a cellular sample in a partition among a plurality of partitions, wherein the cellular sample comprises the nucleic acid sample; and

(c) in the partition, using the at least 1,000 oligonucleotide molecules to barcode the nucleic acid sample from the cellular sample, thereby providing a barcoded nucleic acid molecule derived from the nucleic acid sample in the partition and external to the cellular sample.

2. The method of claim 1, further comprising, prior to (c), subjecting the nucleic acid sample to release from the cellular sample, and subsequently using the at least 1,000 oligonucleotide molecules to barcode the nucleic acid sample.

3. The method of claim 1, wherein the cellular sample is a cell.

4. The method of claim 1, wherein the cellular sample comprises an organelle.

5. The method of claim 1, wherein the cellular sample comprises one or more components of a single cell.

6. The method of claim 1, wherein the partition is a droplet.

7. The method of claim 6, further comprising, subsequent to (c), applying a stimulus to the droplet to release the at least 1,000 oligonucleotide molecules from the bead into the droplet, wherein the bead is degradable upon application of the stimulus.

8. The method of claim 7, wherein the 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.

9. The method of claim 4, wherein (a) comprises providing the bead in an aqueous phase along a first channel of a microfluidic device, and wherein (b) comprises (i) providing a continuous phase along a second channel of the microfluidic device and (ii) bringing the aqueous phase in contact with the continuous phase at a junction of the first channel and the second channel to provide the droplet along a third channel of the microfluidic device.

10. The method of claim 9, further comprising, in (b), combining the at least 1,000 oligonucleotide molecules coupled to the bead, the nucleic acid sample and one or more reagents necessary for amplification of the nucleic acid sample at the junction of the first channel and the second channel to form the droplet along the third channel, wherein the droplet further comprises the one or more reagents.

11. The method of claim 10, wherein the one or more reagents comprises a polymerase.

12. The method of claim 11, wherein the polymerase is incapable of recognizing uracil.

13. The method of claim 1, wherein the partition is a well.

14. The method of claim 1, wherein the at least 1,000 oligonucleotide molecules include at least 10,000 oligonucleotide molecules.

15. The method of claim 1, wherein the at least 1,000 oligonucleotide molecules include at least 100,000 oligonucleotide molecules.

16. The method of claim 1, wherein the at least 1,000 oligonucleotide molecules are releasably attached to the bead.

17. The method of claim 1, wherein the bead is porous.

18. The method of claim 1, further comprising, subsequent to (c), amplifying the nucleic acid sample.

19. The method of claim 18, wherein the nucleic acid sample is amplified with a primer.

20. The method of claim 19, wherein a given oligonucleotide molecule of the at least 1,000 oligonucleotide molecules comprises the primer.

21. The method of claim 18, wherein the amplifying comprises performing reverse transcription on the nucleic acid sample.

22. The method of claim 18, wherein the amplifying comprises performing polymerase chain reaction on the nucleic acid sample.

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

24. The method of claim 23, wherein the region which functions as the primer has a sequence for random priming.

25. The method of claim 1, wherein the at least 1,000 oligonucleotide molecules are coupled to the bead via a chemical cross-linker, a disulfide bond, a covalent bond, or a labile moiety.