# US 11,078,522 B2 · Capsule array devices and methods of use

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- Number: US 11,078,522 B2
- Document ID: US11078522B2
- Status: Granted
- Kind: B2
- Application number: 16/519,863
- Filed: 2019-07-23
- Published: 2020-05-14
- Granted: 2021-08-03
- Earliest claimed: 2012-08-14
- Assignees: 10x Genomics, Inc.
- Applicants: 10X Genomics, Inc.
- Inventors: Benjamin Hindson; Serge Saxonov; Michael Schnall-Levin
- Web page: https://patentgrep.com/patent/US11078522B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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)}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/508: {Rigid containers without fluid transport within}
- B01L 3/523: {with means for closing or opening}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2400/0677: phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- C12Q 2535/122: Massive parallel sequencing
- 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
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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

### Claim 1

A method for sample preparation, comprising:
a) providing a partition comprising a gel bead and a nucleic acid analyte, wherein said gel bead comprises at least 1,000,000 oligonucleotide molecules coupled thereto, wherein said at least 1,000,000 oligonucleotide molecules comprise a common nucleic acid barcode sequence, and wherein said nucleic acid analyte is not coupled to said gel bead;
b) applying a stimulus to release said at least 1,000,000 oligonucleotide molecules from said gel bead, thereby obtaining at least 1,000,000 released oligonucleotide molecules;
c) attaching a released oligonucleotide molecule of said at least 1,000,000 released oligonucleotide molecules to said nucleic acid analyte in said partition; and
d) subjecting said released oligonucleotide molecule attached to said nucleic acid analyte to a nucleic acid extension reaction to yield a barcoded nucleic acid product.

### Claim 2 (depends on 1)

The method of claim 1, wherein said partition is a droplet or a well.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 1)

The method of claim 1, wherein said released oligonucleotide molecule of said at least 1,000,000 released oligonucleotide molecules comprises a region which functions as a primer during said nucleic acid extension reaction.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

The method of claim 11, wherein said released oligonucleotide molecule comprises uracil and said polymerase does not recognize uracil.

### Claim 13 (depends on 1)

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, complementary DNA (cDNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA, cosmid DNA, High Molecular Weight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), short-interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajul body specific RNA (scaRNA), microRNA, double-stranded (dsRNA), ribozyme, riboswitch and viral RNA.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

The method of claim 1, wherein said at least 1,000,000 oligonucleotide molecules are reversibly immobilized to said gel bead.

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 16)

The method of claim 16, wherein nucleic acid analytes of said plurality of nucleic acid analytes attach to individual oligonucleotide molecules of said at least 1,000,000 released oligonucleotide molecules.

### Claim 18 (depends on 16)

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

### Claim 19 (depends on 1)

The method of claim 1, wherein said released oligonucleotide molecule of said at least 1,000,000 released oligonucleotide molecules attaches to said nucleic acid analyte by hybridization.

### Claim 20 (depends on 1)

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

### Claim 21 (depends on 1)

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

### Claim 22 (depends on 21)

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

### Claim 23 (depends on 1)

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