# US 2024/0200057 A1 · Method for Constructing RNA Sequencing Library, Sequencing Method and Kit

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- Number: US 2024/0200057 A1
- Document ID: US20240200057A1
- Status: Published application
- Kind: A1
- Application number: 18/287,887
- Filed: 2021-04-22
- Published: 2024-06-20
- Earliest claimed: 2021-04-22
- Assignees: Bgi Shenzhen
- Applicants: Bgi Shenzhen
- Inventors: Longqi LIU; Xiumei LIN; Quan SHI; Xuyang SHI; Chuanyu LIU; Yaling HUANG; Ya LIU
- Web page: https://patentgrep.com/patent/US20240200057A1

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/25: involving enzymes not classifiable in groups C12Q1/26 {- C12Q1/66}
- C12Q 1/485: {involving kinase}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- G01N 2333/9015: Ligases (6)
- G01N 2333/9128: RNA-directed DNA polymerases, e.g. RT (2.7.7.49)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided in the present application is a method for constructing an RNA sequencing library, a sequencing method and a kit. The construction method comprises: acquiring a single-stranded cDNA, which is a reverse transcription product of mRNA, the 3′-terminal of the single-stranded cDNA including a cDNA tag sequence; cyclizing the single-stranded cDNA to obtain a single-stranded cyclized cDNA; amplifying the single-stranded cyclized cDNA with a primer combination, which is formed by a random primer or a gene-specific primer and a cDNA tag primer, so as to obtain an amplified fragment, the cDNA tag primer being at least a part of the cDNA tag sequence; and performing fragmentation for library construction on the amplified fragment, so as to obtain an RNA sequencing library.

## Claims

### Claim 1

A method for constructing an RNA sequencing library, comprising:
acquiring a single-stranded cDNA, which is a reverse transcription product of mRNA, wherein the 3′-terminal of the single-stranded cDNA comprises a cDNA tag sequence;
cyclizing the single-stranded cDNA to obtain a single-stranded cyclized cDNA;
amplifying the single-stranded cyclized cDNA with a primer combination, which is formed by a random primer or a gene-specific primer and a cDNA tag primer, so as to obtain an amplified fragment, wherein the cDNA tag primer is at least a part of the cDNA tag sequence; and
performing fragmentation for library construction on the amplified fragment, so as to obtain the RNA sequencing library.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the acquiring a single-stranded cDNA, which is a reverse transcription product of mRNA, wherein the 3′-terminal of the single-stranded cDNA comprises a cDNA tag sequence, comprises:
performing reverse transcription on the mRNA, so as to obtain a first strand cDNA;
amplifying the first strand cDNA to obtain a double-stranded cDNA, wherein the 3′ terminal of a second strand cDNA, which is complementary to the first strand cDNA, comprises the cDNA tag sequence, and the cDNA tag sequence comprises a poly(A); and
melting the double-stranded cDNA to obtain the single-stranded cDNA;
preferably, wherein the cDNA tag sequence successively comprises, in a direction from 3′ to 5′, a second PCR adapter, a second cell barcode, a second Unique Molecular Identifier (UMI) and the poly(A).

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The method according to claim 1, wherein the mRNA is derived from a single-cell sample, and the mRNA is a single-cell mRNA;
preferably, wherein preparing the single-cell mRNA with a droplet method, so as to make the single-cell mRNA ligated to a solid support, and more preferably, the solid support is a bead.

### Claim 5

(canceled)

### Claim 6 (depends on 4)

The method according to claim 4, wherein preparing the single-cell mRNA with the droplet method, so as to make the single-cell mRNA ligated to the bead, comprises:
respectively providing a single-cell suspension and the bead, wherein the bead carries a bead tag sequence, and the terminal of the bead tag sequence comprises a poly(dT); and
wrapping the single-cell suspension and the bead into droplets, wherein each droplet comprises one single cell and one bead, and the bead is combined with the poly(A) of the mRNA in the single-cell suspension through the poly(dT), to connect the mRNA in the single-cell suspension to the bead, so as to obtain the single-cell mRNA.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the bead tag sequence successively comprises, in a direction from 5′ to 3′, a first PCR adapter, a first cell barcode, a first UMI and the poly(dT); and correspondingly, the cDNA tag sequence successively comprises, in a direction from 3′ to 5′, a second PCR adapter, a second cell barcode, a second UMI and a poly(A), wherein the second PCR adapter is complementary to the first PCR adapter, the second cell barcode is complementary to the first cell barcode, and the second UMI is complementary to the first UMI.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the 5′ terminal of the single-stranded cDNA comprises a sequence of a TSO primer;
preferably, wherein reverse transcription is performed on the mRNA with a reverse transcriptase and a TSO adapter, so as to obtain the first strand cDNA, wherein the reverse transcriptase has a terminal transferase activity, and the 3′ terminal of the first strand cDNA comprises a complementary sequence of the TSO adapter; and
amplifying the first strand cDNA to obtain the second strand cDNA, and the 5′ terminal of the second strand cDNA comprises the sequence of the TSO primer;
preferably, wherein the sequence of the TSO adapter is SEQ ID NO: 1.

### Claim at position 9

9-10. (canceled)

### Claim 11 (depends on 8)

The method according to claim 8, wherein the reverse transcriptase is selected from an Alpha reverse transcriptase of MGI, a SuperScript™II reverse transcriptase of Invitrogen, Superscript IV of Thermo, or Maxima H Minus of Thermo.

### Claim 12 (depends on 2)

The method according to claim 2, wherein random amplification and/or full-length amplification is performed on the first strand cDNA, so as to obtain the double-stranded cDNA;
preferably, wherein amplifying the first strand cDNA with an adapter amplification primer and a TSO primer, so as to obtain the double-stranded cDNA; or
amplifying the first strand cDNA with the adapter amplification primer, a TSO-random primer and the TSO primer, so as to obtain the double-stranded cDNA;
preferably, wherein the sequence of the adapter amplification primer is SEQ ID NO: 2; the sequence of the TSO primer is SEQ ID NO: 3; and the sequence of the TSO-random primer is SEQ ID NO: 4.

### Claim at position 12

13-14. (canceled)

### Claim 15 (depends on 1)

The method according to claim 1, wherein cyclizing the single-stranded cDNA to obtain a single-stranded cyclized cDNA, comprises:
ligating the single-stranded cDNA into a ring under the action of a cyclization auxiliary sequence and a ligase, so as to obtain a ligated product; and
performing enzyme digestion on the ligated product to digest the single-stranded cDNA, which is not ligated into the ring, so as to obtain the single-stranded cyclized cDNA, wherein
the cyclization auxiliary sequence is complementary to sequences on two terminals of the single-stranded cDNA;
preferably, wherein the cyclization auxiliary sequence is selected from SEQ ID NO: 5.

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The method according to claim 1, wherein the gene-specific primer is a TCR primer for TCR gene amplification and/or a BCR primer for BCR gene amplification.

### Claim 18 (depends on 1)

The method according to claim 1, wherein the cDNA tag primer is a poly(A) primer, preferably SEQ ID NO: 6.

### Claim 19 (depends on 1)

The method according to claim 1, wherein the performing fragmentation for library construction on the amplified fragment, so as to obtain an RNA sequencing library, comprises:
adding a library adapter to the amplified fragment, so as to obtain the RNA sequencing library;
preferably, wherein enzyme digestion fragmentation is performed on the amplified fragment, so as to obtain digested fragments; and
terminal repair, A tailing addition and library adapter ligation are successively performed on the digested fragments, so as to obtain the RNA sequencing library.

### Claim 20

(canceled)

### Claim 21 (depends on 19)

The method according to claim 19, wherein after library adapter ligation is performed, the method further comprises performing PCR amplification on the ligated product of the library adapter, so as to obtain the RNA sequencing library;
preferably wherein the library adapter is an adapter of an MGI sequencing platform or an adapter of an Illumina sequencing platform.

### Claim 22

(canceled)

### Claim 23

A kit for an RNA library construction, comprising: a cyclization auxiliary sequence, a DNA ligase, a cDNA tag primer, and at least one of the following primers: (a) a random primer; (b) a TCR primer; or (c) a BCR primer.

### Claim 24 (depends on 23)

The kit according to claim 23, further comprising an RNA reverse transcription reagent;
preferably, wherein the RNA reverse transcription reagent comprises a reverse transcriptase; and the reverse transcriptase is a reverse transcriptase having terminal transferase activity;
preferably, wherein the reverse transcriptase is selected from an Alpha reverse transcriptase of MGI, a SuperScript™II reverse transcriptase of Invitrogen, Superscript IV of Thermo, or Maxima H Minus;
preferably, wherein the RNA reverse transcription reagent further comprises a TSO adapter;
preferably, wherein the sequence of the TSO adapter is SEQ ID NO: 1.

### Claim at position 23

25-28. (canceled)

### Claim 29 (depends on 23)

The kit according to claim 23, further comprising a TSO primer and an adapter amplification primer;
preferably, wherein the sequence of the adapter amplification primer is SEQ ID NO: 2;
and the sequence of the TSO primer is SEQ ID NO: 3;
preferably, wherein the kit further comprises a TSO-random primer;
more preferably, wherein the sequence of the TSO-random primer is SEQ ID NO: 4.

### Claim at position 25

30-32. (canceled)

### Claim 33 (depends on 23)

The kit according to claim 23, wherein the cyclization auxiliary sequence is SEQ ID NO: 5;
preferably, wherein the cDNA tag primer is a poly(A) primer, more preferably, wherein a sequence of the cDNA tag primer is SEQ ID NO:6;
preferably, the kit further comprises at least one of an exonuclease or a library adapter;
more preferably, wherein the exonuclease is selected from an exonuclease I or exonuclease III;
preferably, wherein the library adapter is an adapter of an MGI sequencing platform or an adapter of an Illumina sequencing platform; more preferably, wherein the MGI sequencing platform is selected from a bubble adapter; and an adapter of the Illumina sequencing platform is selected from P5 and P7 adapters.

### Claim at position 27

34-39. (canceled)

### Claim 40 (depends on 23)

The kit according to claim 23, wherein the DNA ligase is selected from a T4 DNA ligase.

### Claim 41 (depends on 23)

The kit according to claim 23, further comprising a solid support, wherein the solid support is provided with a support tag sequence; the cDNA tag primer is complementary to at least a part of the support tag sequence; and preferably, the solid support is a bead, and the support tag sequence is a bead tag sequence;
preferably wherein the support tag sequence successively comprises, in a direction from 5′ to 3′, a first PCR adapter, a first cell barcode, a first UMI and the poly(dT).

### Claim 42

(canceled)

### Claim 43 (depends on 1)

A method for sequencing an RNA library, comprising:
constructing an RNA sequencing library with the method for constructing an RNA sequencing library according to claim 1, and
performing sequencing on the RNA sequencing library.