# US 10,870,847 B2 · Methods and kits for nucleic acid sample preparation for sequencing

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- Number: US 10,870,847 B2
- Document ID: US10870847B2
- Status: Granted
- Kind: B2
- Application number: 15/945,557
- Filed: 2018-04-04
- Published: 2019-03-14
- Granted: 2020-12-22
- Earliest claimed: 2012-11-02
- Assignees: ArcherDX, Inc.
- Applicants: ArcherDX, LLC
- Inventors: Patrick Finn; Greg Patton; Hongbo Liu
- Web page: https://patentgrep.com/patent/US10870847B2

## CPC

- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The present disclosure relates to methods and kits for DNA library construction, particularly for consistent and reproducible DNA sequencing.

## Claims

### Claim 1

A method for DNA library construction, comprising:
modifying nucleic acid fragments; and
ligating nucleic acid adaptors to the modified nucleic acid fragments;
wherein one or more of the reactions are rehydrated from a lyophilized form in a reaction volume comprising the nucleic acid fragments.

### Claim 2

A method for DNA library construction, comprising:
modifying DNA fragments with a polymerase and a polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with a (3′→5′ exo-) polymerase in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA fragments in a third reaction mixture comprising a nucleic acid ligase;
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments.

### Claim 3 (depends on 2)

The method of claim 2, wherein at least the first reaction mixture is rehydrated from a lyophilized form.

### Claim 4 (depends on 3)

The method of claim 3, wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.

### Claim 5 (depends on 4)

The method of claim 4, wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.

### Claim 6 (depends on 3)

The method of claim 3, wherein the lyophilized form comprises T4 DNA polymerase, T4 DNA polynucleotide kinase, deoxynucleotide triphosphates, a source of magnesium, and a buffer.

### Claim 7 (depends on 3)

The method of claim 3, wherein the DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized first reaction mixture.

### Claim 8 (depends on 2)

The method of claim 2, wherein at least the second reaction mixture is rehydrated from a lyophilized form.

### Claim 9 (depends on 8)

The method of claim 8, wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.

### Claim 10 (depends on 9)

The method of claim 9, wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.

### Claim 11 (depends on 8)

The method of claim 8, wherein the lyophilized form comprises Klenow (3′→5′ exo-), a source of magnesium, a source of sodium, dATP, and a buffer.

### Claim 12 (depends on 8)

The method of claim 8, wherein the end repaired DNA fragments in the reaction volume are added to a single use reaction tube containing the lyophilized second reaction mixture.

### Claim 13 (depends on 2)

The method of claim 2, wherein at least the third reaction mixture is rehydrated from a lyophilized form.

### Claim 14 (depends on 13)

The method of claim 13, wherein the lyophilized form is one or more lyophilized pellets, and wherein each lyophilized pellet has a diameter of from about 0.3 cm to about 1.0 cm.

### Claim 15 (depends on 14)

The method of claim 14, wherein the lyophilized pellet is lyophilized from an approximately 15 μl to approximately 300 μl volume.

### Claim 16 (depends on 13)

The method of claim 13, wherein the lyophilized form comprises T4 DNA ligase, a source of magnesium, ATP, and a buffer.

### Claim 17 (depends on 13)

The method of claim 13, wherein the A-tailed DNA in the reaction volume are added to a single use reaction tube containing the lyophilized third reaction mixture.

### Claim 18 (depends on 2)

The method of claim 2, wherein at least two of the first reaction mixture, second reaction mixture, and third reaction mixture are rehydrated from lyophilized form.

### Claim 19

A method of for DNA library construction, comprising:
modifying DNA fragments with T4 DNA polymerase and T4 polynucleotide kinase in a first reaction mixture sufficient for end repair of the DNA fragments;
contacting the end repaired DNA fragments with Klenow (3′→5′ exo-) in a second reaction mixture sufficient for A-tailing the end-repaired DNA fragments;
ligating DNA adaptors to the A-tailed DNA in a third reaction mixture comprising T4 DNA ligase,
wherein one or more of the first reaction mixture, the second reaction mixture, and the third reaction mixture are rehydrated from a lyophilized form in a reaction volume comprising the DNA fragments.