# US 2016/0257984 A1 · Processes and Systems for Preparation of Nucleic Acid Sequencing Libraries and Libraries Prepared Using Same

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- Number: US 2016/0257984 A1
- Document ID: US20160257984A1
- Status: Published application
- Kind: A1
- Application number: 14/990,276
- Filed: 2016-01-07
- Published: 2016-09-08
- Earliest claimed: 2015-01-12
- Assignees: 10x Genomics, Inc.
- Applicants: 10X Genomics, Inc.
- Inventors: Paul Hardenbol; Pranav Patel; Benjamin Hindson; Paul William Wyatt; Keith Bjornson; Indira Wu; Kamila Belhocine
- Web page: https://patentgrep.com/patent/US20160257984A1

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C40B 20/04: Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
- B01J 2219/00547: Bar codes
- B01J 2219/00572: Chemical means
- B01J 2219/00722: Nucleotides
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

This disclosure provides methods for preparing a sequencing library including the steps of providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads including oligonucleotide adapter sequence segments; amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence including occasional dUTPs; and excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

## Claims

### Claim 1

A method of preparing a sequencing library comprising the steps of:
(a) providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, and a plurality of beads comprising oligonucleotide adapter sequence segments;
(b) amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence comprising occasional dUTPs; and
(c) excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence to provide a sequencing library.

### Claim 2 (depends on 1)

The method of claim 1, further comprising a step (d) of amplifying the nicked complementary nucleic acid sequence, and a step (e) of extending the sequence of the amplified nucleic acid sequence using a nucleic acid extension means.

### Claim 3 (depends on 1)

The method of claim 1, wherein the steps are performed in a single reaction.

### Claim 4 (depends on 1)

The method of claim 1, wherein the plurality of beads is a pooled bead population.

### Claim 5 (depends on 4)

The method of claim 4, wherein the beads of the pooled bead population are co-partitioned with one or more of the components listed in step (a), and wherein the partition optionally comprises a droplet in an emulsion.

### Claim 6 (depends on 1)

The method of claim 1, wherein the beads comprise degradable beads selected from chemically degradable beads, photodegradable beads and thermally degradable beads.

### Claim 7 (depends on 6)

The method of claim 6, wherein the beads comprise chemically reducible cross-linkers.

### Claim 8 (depends on 7)

The method of claim 7, wherein the chemically reducible cross-linkers comprise disulfide linkages.

### Claim 9 (depends on 1)

The method of claim 1, wherein the amplification in step (b) is isothermal.

### Claim 10 (depends on 1)

The method of claim 1, wherein the polymerase is phi29 DNA polymerase.

### Claim 11 (depends on 2)

The method of claim 2, wherein the nucleic acid extension means is selected from the group consisting of a ligating enzyme, a nucleic acid extension enzyme and a transposase.

### Claim 12 (depends on 11)

The method of claim 11 wherein the library of amplified nucleic acid sequences comprises single stranded DNA and the ligating enzyme comprises an ATP independent enzyme.

### Claim 13 (depends on 12)

The method of claim 12, wherein the ATP independent enzyme comprises thermostable 5′ App DNA/RNA ligase.

### Claim 14 (depends on 11)

The method of claim 11, wherein the ligating enzyme comprises a topoisomerase.

### Claim 15 (depends on 14)

The method of claim 14, wherein the topoisomerase is topoisomerase I.

### Claim 16 (depends on 11)

The method of claim 11, wherein the ligating enzyme comprises T4 DNA ligase.

### Claim 17

A method of preparing a barcode sequencing library, comprising:
(a) providing a template nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising enzyme, a nucleic acid extension means and a plurality of beads comprising oligonucleotide barcode sequence segments;
(b) amplifying the template nucleic acid with the polymerase, dNTPs, dUTP and random hexamer to provide a complementary nucleic acid sequence comprising occasional dUTPs; and
(c) excising the incorporated dUTPs with the dUTP excising enzyme to provide nicks in the complementary nucleic acid sequence;
(d) amplifying the nicked complementary nucleic acid sequence to provide a library of amplified nucleic acid sequences; and
(e) releasing the barcode sequence segments from the pooled bead population; and
(f) extending the sequence of the amplified nucleic acid sequences using the barcode sequence segments and the nucleic acid extension means to provide a barcode library or alternatively, ligating the barcode sequence segments, using a nucleic acid ligating enzyme, to the library of amplified nucleic acid sequences to provide a barcode library.

### Claim 18 (depends on 17)

The method of claim 17, wherein the steps are performed in a single reaction.

### Claim 19 (depends on 17)

The method of claim 17, wherein the plurality of beads is a pooled bead population.

### Claim 20 (depends on 19)

The method of claim 19, wherein the beads of the pooled bead population are co-partitioned with one or more of the components listed in step (a), and wherein the partition optionally comprises a droplet in an emulsion.

### Claim 21 (depends on 17)

The method of any of claim 17, wherein the beads comprise degradable beads selected from chemically degradable beads, photodegradable beads and thermally degradable beads.

### Claim 22 (depends on 21)

The method of claim 21, wherein the beads comprise chemically reducible cross-linkers.

### Claim 23 (depends on 22)

The method of claim 22, wherein the chemically reducible cross-linkers comprise disulfide linkages.

### Claim 24 (depends on 17)

The method of claim 17, wherein the amplification in step (b) is isothermal.

### Claim 25 (depends on 17)

The method of claim 17, wherein the polymerase is phi29 DNA polymerase.

### Claim 26 (depends on 17)

The method of claim 17, wherein the nucleic acid extension means is selected from the group consisting of a ligating enzyme, a nucleic acid extension enzyme and a transposase.

### Claim 27 (depends on 26)

The method of claim 26 wherein the library of amplified nucleic acid sequences comprises single stranded DNA and the ligating enzyme comprises an ATP independent enzyme.

### Claim 28 (depends on 27)

The method of claim 27, wherein the ATP independent enzyme comprises thermostable 5′ App DNA/RNA ligase.

### Claim 29 (depends on 26)

The method of claim 26, wherein the ligating enzyme comprises a topoisomerase.

### Claim 30 (depends on 29)

The method of claim 29, wherein the topoisomerase is topoisomerase I.

### Claim 31 (depends on 26)

The method of claim 26, wherein the ligating enzyme comprises T4 DNA ligase.

### Claim 32 (depends on 17)

The method of claim 17, wherein the barcode sequence segments comprise at least 4 nucleotides at least 10 nucleotides or at least 20 nucleotides.

### Claim 33 (depends on 17)

The method of claim 17, wherein the barcode sequence segments comprise at least 1000 different barcode sequence segments.

### Claim 34 (depends on 17)

The method of claim 17, wherein at least 1,000,000 oligonucleotide molecules are attached to each bead.

### Claim 35 (depends on 19)

The method of claim 19, wherein the pooled bead population comprises at least 10 different bead populations.

### Claim 36 (depends on 19)

The method of claim 19, wherein the pooled bead population comprises at least 100 different bead populations.

### Claim 37 (depends on 19)

The method of claim 19, wherein the pooled bead population comprises at least 500 different bead populations.

### Claim 38 (depends on 17)

The method of claim 17, wherein the oligonucleotide barcode sequence segments comprise at least one functional sequence.

### Claim 39 (depends on 38)

The method of claim 38, wherein the functional sequence is selected from an adapter, a primer sequence, a primer annealing sequence, an attachment sequence, and a sequencing primer sequence.

### Claim 40 (depends on 38)

The method of claim 38, wherein the functional sequence is sequestered and releasable in a releasing step comprising a stimulus selected from the list consisting of thermal increase and chemical cleavage.

### Claim 41 (depends on 40)

The method of claim 40, wherein the releasing step comprises degrading at least a portion the beads of the bead population comprising oligonucleotide barcode sequence segments.

### Claim 42 (depends on 41)

The method of claim 41, wherein degrading the beads comprises cleaving a chemical linkage comprising a disulfide bridge linkage between the barcode sequence segments and the bead, and the releasing step comprises exposing the beads to a reducing agent.

### Claim 43 (depends on 42)

The method of claim 42, wherein the reducing agent comprises a reducing agent selected from the group consisting of DTT and TCEP.