# US 10,870,848 B2 · Methods for preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample and compositions for practicing the same

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- Number: US 10,870,848 B2
- Document ID: US10870848B2
- Status: Granted
- Kind: B2
- Application number: 15/746,781
- Filed: 2016-09-15
- Published: 2019-01-10
- Granted: 2020-12-22
- Earliest claimed: 2015-09-15
- Assignees: Takara Bio USA, Inc.
- Applicants: Takara Bio USA, Inc.
- Inventors: Cynthia Chang; Magnolia Bostick
- Web page: https://patentgrep.com/patent/US10870848B2

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- 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)
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- 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
- 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)

## Abstract

Methods of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample are provided. Aspects of the methods include combining the RNA sample with a first strand cDNA primer and a template switch oligo-nucleotide under first strand cDNA synthesis conditions, where one of the first strand cDNA primer and the template switch oligo-nucleotide includes a first post-tagmentation amplification primer binding domain. The resultant product is subjected to amplification conditions sufficient to produce a double stranded cDNA, which is then tagmented with a transposome that includes a second post-tagmentation amplification primer binding domain. The tagmented sample is then subjected to amplification conditions using first and second post-tagmentation amplification primers that include sequencing platform adapter constructs to produce a NGS library. Aspects of the invention further include compositions produced by the methods and kits that find use in practicing the methods.

## Claims

### Claim 1

A method of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample, the method comprising:
(a) combining:
a RNA sample;
a first strand complementary deoxyribonucleic acid (cDNA) primer comprising a first pre-tagmentation amplification primer binding domain;
a template switch oligonucleotide comprising a 3′ hybridization domain and a 5′ second pre-tagmentation amplification primer binding domain;
a reverse transcriptase; and
dNTPs;
in a reaction mixture under conditions sufficient to produce a double stranded product nucleic acid comprising a template mRNA and the template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein only one of the first strand cDNA primer and the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain; and
(b) contacting the product nucleic acid with first and second pre-tagmentation amplification primers complementary to the first and second pre-tagmentation amplification primer binding domains under amplification conditions sufficient to produce a product double stranded cDNA;
(c) tagmenting the product double stranded cDNA with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon end domain and a second post-tagmentation amplification primer binding domain to produce a tagmented sample; and
(d) amplifying the tagmented sample by contacting the tagmented sample with:
a first post-tagmentation primer comprising a first post-tagmentation amplification primer domain complementary to the first post-tagmentation amplification primer binding domain; and
a second primer comprising a second post-tagmentation amplification primer domain complementary to the second post-tagmentation amplification primer binding domain.

### Claim 2 (depends on 1)

The method according to claim 1, wherein
the first post-tagmentation primer further comprises a first NGS indexing domain and a first NGS adapter domain; and
the second post-tagmentation primer further comprises a second NGS indexing domain and a second NGS adapter domain.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the RNA sample comprises messenger RNA and the method comprises producing the NGS library from mRNA.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the first strand cDNA primer comprises the first post-tagmentation amplification primer binding domain.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the template switch oligonucleotide comprises the first post-tagmentation amplification primer binding domain.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the first and second pre-tagmentation amplification primer binding domains are identical and the first and second pre-tagmentation amplification primers are identical.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the transposase comprises a Tn5 transposase.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the transposon end domain comprises a Tn5 transposon end domain.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the method further comprises pooling the double stranded product cDNA with a second double stranded product cDNA to produce a pooled cDNA sample, and then tagmenting the pooled cDNA sample.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the RNA sample is one that is produced from a single cell.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the method further comprises subjecting the NGS library to an NGS protocol.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the method further comprises quantitating one or more RNA species of the RNA sample.

### Claim 13 (depends on 4)

The method according to claim 4, wherein the first strand cDNA primer comprises an oligodT domain 3′ of the first pre-tagmentation amplification primer binding domain.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the first strand cDNA primer further comprises a source barcode domain.

### Claim 15 (depends on 1)

The method according to claim 1, wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs.

### Claim 16 (depends on 15)

The method according to claim 15, wherein each of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs.

### Claim 17 (depends on 15)

The method according to claim 15, wherein the one or more nucleotide analogs is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, linkage modifications, 5′ and/or 3′ end modifications, one or more fluorescently labeled nucleotides, and combinations thereof.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the one or more nucleotide analogs comprises a 5′ end modification.

### Claim 19 (depends on 1)

The method according to claim 1, wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation primers comprises a 5′ polymerase blocking modification.

### Claim 20 (depends on 19)

The method according to claim 19, wherein each of the first strand cDNA primer and pre-tagmentation primers comprises a 5′ polymerase blocking modification.

### Claim 21 (depends on 19)

The method according to claim 19, wherein the 5′ polymerase blocking modification is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, and combinations thereof.

### Claim 22 (depends on 1)

The method according to claim 1, wherein the 3′ hybridization domain comprises a homonucleotide stretch.

### Claim 23 (depends on 1)

The method according to claim 1, wherein the 3′ hybridization domain comprises a heteronucleotide stretch.