# US 11,441,179 B2 · Methods and systems for processing polynucleotides

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,441,179 B2
- Document ID: US11441179B2
- Status: Granted
- Kind: B2
- Application number: 16/844,141
- Filed: 2020-04-09
- Published: 2021-01-07
- Granted: 2022-09-13
- Earliest claimed: 2012-08-14
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Christopher Hindson; Michael Schnall-Levin; Andrew Price; Paul Hardenbol; Yuan Li
- Web page: https://patentgrep.com/patent/US11441179B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6869: Methods for sequencing
- 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

## 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

### Claim 1

A method, comprising:
(a) providing, in a first plurality of wells, a plurality of beads comprising a first plurality of nucleic acid molecules, wherein said first plurality of nucleic acid molecules comprises a plurality of first partial barcode sequences, wherein said plurality of first partial barcode sequences differ across said first plurality of wells;
(b) pooling said plurality of beads from said first plurality of wells, thereby generating a pooled population of beads;
(c) partitioning, in a second plurality of wells, said pooled population of beads and a second plurality of nucleic acid molecules, wherein a well of said second plurality of wells comprises (i) a bead of said pooled population of beads, wherein said bead comprises a first nucleic acid molecule of said first plurality of nucleic acid molecules and (ii) a second nucleic acid molecule of said second plurality of nucleic acid molecules;
wherein said second nucleic acid molecule comprises a poly-T sequence, a random sequence, or a targeted primer sequence; and
(d) providing conditions sufficient to permit coupling of said first nucleic acid molecule to said second nucleic acid molecule, thereby generating a barcoded bead comprising a nucleic acid barcode molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein said first plurality of wells comprises M wells and said second plurality of wells comprises N wells, wherein M≥96 and N≥96.

### Claim 3 (depends on 1)

The method of claim 1, wherein said plurality of beads comprises a plurality of linkage moieties, wherein said first plurality of nucleic acid molecules is attached to said beads via said plurality of linkage moieties.

### Claim 4 (depends on 3)

The method of claim 3, wherein said plurality of linkage moieties is coupled to a plurality of nucleic acid anchor molecules.

### Claim 5 (depends on 4)

The method of claim 4, further comprising, prior to (a), coupling said first plurality of nucleic acid molecules to said plurality of nucleic acid anchor molecules using a plurality of complementary nucleic acid molecules, wherein a complementary nucleic acid molecule of said plurality of complementary nucleic acid molecules comprises (i) a first sequence complementary to at least a portion of a nucleic acid anchor molecule of said plurality of nucleic acid anchor molecules and (ii) a second sequence complementary to at least a portion of a first nucleic acid molecule of said first plurality of nucleic acid molecules.

### Claim 6 (depends on 5)

The method of claim 5, wherein said first nucleic acid molecule is hybridized to said second sequence prior to said coupling.

### Claim 7 (depends on 6)

The method of claim 6, further comprising hybridizing said at least said portion of said nucleic acid anchor molecule to said first sequence.

### Claim 8 (depends on 7)

The method of claim 7, further comprising ligating said first nucleic acid molecule to said nucleic acid anchor molecule.

### Claim 9 (depends on 8)

The method of claim 8, further comprising removing said first sequence from said nucleic acid anchor molecule.

### Claim 10 (depends on 9)

The method of claim 9, wherein said removing is performed via denaturation.

### Claim 11 (depends on 4)

The method of claim 4, wherein said barcoded bead comprises the structure: bead-said nucleic acid anchor molecule-said first partial barcode sequence-said poly-T sequence, said random sequence or said targeted primer sequence.

### Claim 12 (depends on 4)

The method of claim 4, wherein said plurality of nucleic acid anchor molecules comprises an adapter sequence comprising a primer sequence, a primer binding sequence, a partial primer sequence or a partial primer binding sequence.

### Claim 13 (depends on 12)

The method of claim 12, wherein (i) said primer sequence is a sequencing primer sequence; (ii) said primer binding sequence is a sequencing primer binding sequence; (iii) said partial primer sequence is a partial sequencing primer sequence, or (iv): said partial primer binding sequence is a partial sequencing primer binding sequence.

### Claim 14 (depends on 4)

The method of claim 4, wherein said plurality of nucleic acid anchor molecules comprises a sequence configured to attach to a flow cell of a sequencer.

### Claim 15 (depends on 4)

The method of claim 4, wherein a linkage moiety of said plurality of linkage moieties is coupled to a bead of said plurality of beads via a labile bond.

### Claim 16 (depends on 15)

The method of claim 15, wherein said labile bond is a thermally labile bond, a photolabile bond, or a chemically labile bond.

### Claim 17 (depends on 16)

The method of claim 16, wherein said labile bond comprises an ester linkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfone linkage, a silyl ether linkage, a glycosidic linkage, or a peptide linkage.

### Claim 18 (depends on 1)

The method of claim 1, further comprising, prior to (a), providing conditions sufficient to permit joining of a first partial barcode sequence of said plurality of first partial barcode sequences to an additional partial barcode sequence.

### Claim 19 (depends on 1)

The method of claim 1, wherein said first nucleic acid molecule comprises a plurality of partial barcode sequences.

### Claim 20 (depends on 19)

The method of claim 19, further comprising, prior to (a):
(i) partitioning an initial plurality of beads comprising a plurality of nucleic acid anchor molecules and an initial plurality of nucleic acid molecules comprising said plurality of first partial barcode sequences into a first initial plurality of wells;
(ii) providing conditions sufficient to permit joining of said initial plurality of nucleic acid molecules to said plurality of nucleic acid anchor molecules, thereby generating a plurality of bead-oligonucleotide constructs;
(iii) pooling said plurality of bead-oligonucleotide constructs, thereby generating an initial pooled population of beads;
(iv) partitioning said initial pooled population of beads and a second initial plurality of nucleic acid molecules comprising at least one additional partial barcode sequence into a second initial plurality of wells; and
(v) providing conditions sufficient to permit joining of said second initial plurality of nucleic acid molecules to bead-oligonucleotide constructs of said initial pooled population of beads of (iv), thereby generating said plurality of beads comprising said first plurality of nucleic acid molecules in (a).

### Claim 21 (depends on 20)

The method of claim 20, wherein (ii) or (v) is completed using a nucleic acid splint molecule.

### Claim 22 (depends on 1)

The method of claim 1, wherein, after (d), said barcoded bead comprises a plurality of nucleic acid barcode molecules comprising a common nucleic acid barcode sequence.

### Claim 23 (depends on 1)

The method of claim 1, wherein barcode sequences of said plurality of first partial barcode sequences are from 4 to 20 nucleic acid bases in length.

### Claim 24 (depends on 1)

The method of claim 1, wherein said plurality of beads is a plurality of gel beads.

### Claim 25 (depends on 24)

The method of claim 24, wherein gel beads of said plurality of gel beads are degradable upon application of a stimulus.

### Claim 26 (depends on 25)

The method of claim 25, wherein said stimulus comprises a thermal stimulus, a chemical stimulus, a biological stimulus, or a photo-stimulus.

### Claim 27 (depends on 25)

The method of claim 25, wherein said stimulus comprises a reducing agent.

### Claim 28 (depends on 1)

The method of claim 1, wherein said second plurality of nucleic acid molecules comprises a plurality of second partial barcode sequences, wherein said plurality of second partial barcode sequences differ across said second plurality of wells.

### Claim 29 (depends on 1)

The method of claim 1, wherein said first nucleic acid molecule comprises only one partial barcode sequence.

### Claim 30 (depends on 1)

The method of claim 1, wherein said conditions sufficient to permit coupling of said first nucleic acid molecule to said second nucleic acid molecule comprises performing a ligation reaction or a nucleic acid extension reaction.

### Claim 31 (depends on 1)

The method of claim 1, wherein said plurality of first partial barcode sequences comprises at least 100,000 different barcode sequences.

### Claim 32 (depends on 1)

The method of claim 1, wherein said first nucleic acid molecule comprises a linking sequence, wherein said second nucleic acid molecule comprises a complementary sequence to said linking sequence, and wherein (d) comprises hybridizing said linking sequence to said complementary sequence.

### Claim 33 (depends on 32)

The method of claim 32, further comprising, subjecting said first nucleic acid molecule hybridized to said second nucleic acid molecule to a nucleic acid extension reaction.

### Claim 34 (depends on 32)

The method of claim 32, wherein said linking sequence is 2-10 nucleotides in length.

### Claim 35 (depends on 32)

The method of claim 32, wherein said linking sequence is greater than 10 nucleotides in length.

### Claim 36 (depends on 1)

The method of claim 1, wherein said first nucleic acid molecule is partially double-stranded comprising a single-stranded overhang sequence, and wherein said second nucleic acid is partially double-stranded and comprises a complementary sequence that is complementary to said single-stranded overhang sequence.

### Claim 37 (depends on 1)

The method of claim 1, wherein said barcoded bead comprises a unique molecular identifier sequence.

### Claim 38 (depends on 1)

The method of claim 1, wherein said nucleic acid barcode molecule comprises a uracil base.

### Claim 39 (depends on 1)

The method of claim 1, wherein said barcoded bead comprises at least 1,000,000 oligonucleotides coupled thereto, wherein said at least 1,000,000 oligonucleotides comprises said nucleic acid barcode molecule.