# US 11,993,805 B2 · Methods, compositions, and kits for preparing nucleic acid libraries

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- Number: US 11,993,805 B2
- Document ID: US11993805B2
- Status: Granted
- Kind: B2
- Application number: 17/869,595
- Filed: 2022-07-20
- Published: 2023-04-20
- Granted: 2024-05-28
- Earliest claimed: 2017-04-17
- Assignees: Genefirst Ltd.
- Applicants: Genefirst Ltd.
- Inventors: Guoliang Fu; Thomas Dunwell
- Web page: https://patentgrep.com/patent/US11993805B2

## CPC

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

## Abstract

This invention relates to methods, compositions and kits for extending a polynucleotide and for preparing sequencing library of polynucleotides involving generating modified target polynucleotide on an adaptor template oligonucleotide and tagging one or two strands of a target sequence. The sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.

## Claims

### Claim 1

An adaptor template oligonucleotide (ATO) for extending polynucleotides comprising:
(a) a 3′ random sequence of 3 to 36 ‘N’ bases;
(b) a 3′ end with a blocker, which renders ATO non-extendible;
(c) a universal sequence, 5′ to the random sequence;
(d) modified nucleotides or linkages which render the ATO resistant to 3′ exonuclease cleavage; and
(e) a uracil nucleotide which renders the ATO degradable.

### Claim 2 (depends on 1)

The adaptor template oligonucleotide (ATO) according to claim 1 wherein the modified nucleotides or linkages which render the ATO resistant to 3′ exonuclease cleavage are phosphorothioate linkages.

### Claim 3 (depends on 1)

The adaptor template oligonucleotide (ATO) according to claim 1 wherein the universal sequence comprises a sequence capable of acting as an RNA polymerase promoter.

### Claim 4 (depends on 1)

The adaptor template oligonucleotide (ATO) according to claim 1 wherein the ATO comprises a 5′ stem portion sequence which is complementary or partially complementary to part of or all of the universal sequence, and is therefore capable of forming a stem-loop structure.

### Claim 5 (depends on 4)

The adaptor template oligonucleotide according to claim 4, wherein the ATO comprises in 5′ to 3′ order: a 5′ stem portion, an RNA polymerase sequence, a priming site sequence, a single stranded overhanging 3′ random sequence and a 3′ end with a blocker.

### Claim 6 (depends on 4)

The adaptor template oligonucleotide according to claim 4 wherein the stem-loop structure contains a non-copiable linkage.

### Claim 7 (depends on 6)

The adaptor template oligonucleotide according to claim 6, wherein the non-copiable linkage is a C3 Spacer, a triethylene glycol spacer, an 18-atom hexa-ethyleneglycol spacer, or 1′,2′-Dideoxyribose (dSpacer).

### Claim 8 (depends on 1)

The adaptor template oligonucleotide according to claim 1 wherein the 3′ end is blocked with a moiety selected from the group consisting of at least one ribonucleotide, at least one deoxynucleotide, a C3 spacer, a phosphate, a dideoxynucleotide, an amino group, and an inverted deoxythymidine.

### Claim 9 (depends on 1)

A kit for generating a library of polynucleotides comprising an adaptor template oligonucleotide (ATO) according to claim 1, a polymerase having 3′ exonuclease activity and primers compatible to an NGS platform.