CN115874292A - Method for removing joint self-ligation product in sequencing process of small RNA library - Google Patents

Abstract

The invention discloses a method for removing a linker self-ligation product in a sequencing library construction process. Specifically, provided is a method for cleaving a linker self-ligation product using an endonuclease, the method comprising the step of mixing the endonuclease and the linker self-ligation product: wherein, the joint self-ligation product has an endonuclease recognition site, and the two joints of the site have a part respectively; the endonuclease can recognize the enzyme cutting site and cut; optionally, the adaptor self-ligation product is generated during construction of an RNA sequencing library; optionally, the linkers are 5 'linkers and 3' linkers used during RNA sequencing library construction.

Description

Method for removing joint self-ligation product in sequencing process of small RNA library

Technical Field

The invention relates to the field of biotechnology, in particular to the field of gene sequencing. In particular, the invention relates to methods for removing adaptor self-ligation products during sequencing library construction. More specifically, the invention relates to a method for removing a linker self-ligation product in a sequencing library construction process by using endonuclease, a method for constructing a sequencing library based on a small RNA sample, a kit for constructing the small RNA sequencing library and a method for determining sequence information of a small RNA molecule.

Background

Small RNAs are a large class of regulatory molecules in organisms, including miRNA, siRNA, piRNA, snRNA, snorRNA, srRNA and the like, and play important regulatory roles in organisms. Several studies have been successful in recent years to use the lineage of small RNAs as markers for specific disease diagnosis. In the future, the detection of small RNA will be widely applied to early diagnosis, typing and individualized detection treatment of diseases. Common small RNA quantitative detection techniques include deep sequencing techniques, chip techniques, and qRT-PCR techniques. The latter two require the synthesis of specific probes and therefore can only detect small RNAs of known species. The high-throughput sequencing technology has the advantages of high flux, high sensitivity, no need of any previous sequence information and secondary structure information, capability of discovering new small RNA molecules and the like, and is widely applied to the field of small RNA research. The library construction method for deep sequencing of small RNA is mainly a two-step joint connection reverse transcription method. The method is suitable for the construction of any RNA deep sequencing library suitable for ligation reactions, including disrupted long transcript RNA, such as small RNA sequencing, CLIP sequencing, RIP sequencing, GRO sequencing and the like.

Due to the technical limitation of small RNA sequencing, the existing library construction method of small RNA or RNA fragments is still difficult to detect the small RNA in a trace sample (less than 100ng of total RNA or 1ng of small RNA). The step of library construction of small RNAs or RNA fragments first requires the ligation of a3 'linker sequence to the 3' end of the small RNA, followed by the ligation of a5 'linker sequence to the 5' end of the small RNA or RNA fragment. This fraction was subjected to reverse transcription and PCR amplification to obtain a library for deep sequencing. During the ligation reaction, a ligation reaction between excess 5 'and 3' linkers occurs, producing a waste byproduct. For ligation reactions that start with very low amounts of small RNA or RNA fragments, the ligation between the 5 'and 3' linkers produces a significant majority of by-products that severely hinder subsequent PCR amplification of the library.

In the existing solution, the cleartag technology of Trilink company adds special modification to the ends of the 5 'linker and the 3' linker, so that the linker self-ligation product cannot be effectively extended by reverse transcriptase to remove the linker self-ligation contamination, while Wu Li just and the like of Shanghai institute of Life sciences of China remove the linker contamination by a method of cutting the linker self-ligation product by Cas9 enzyme, and the corresponding sequencing method is CAS-seq. Among these methods, the cleartag technology is costly and only partially decontaminates, whereas the CAS-seq technology, which uses CAS9 enzyme, may have off-target effect, cleaves small RNA inserts, and is costly and complex in experimental operation.

Therefore, how to simply and efficiently remove the byproducts generated by the connection between the 5 'and 3' linkers is the key to realize the library establishment of trace small RNAs or RNA fragments.

Disclosure of Invention

Specifically, the present invention solves the above technical problems in the prior art by the following technical solutions.

1. A method for cleaving a linker self-ligation product using an endonuclease, the method comprising mixing the endonuclease and the linker self-ligation product, wherein the linker self-ligation product comprises a recognition site for the endonuclease at which the endonuclease cleaves;

optionally, the adaptor self-ligation product is generated during construction of a sequencing library;

optionally, the adaptor self-ligation product is formed by ligation of a5 'end adaptor and a3' end adaptor used in the sequencing library construction process;

optionally, the 5 'end linker and the 3' end linker each contain a partial sequence of the recognition site, and when the linker self-ligation product is formed, a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker;

optionally, the sequencing library is an RNA sequencing library.

2. A method of removing 5 'end linker and 3' end linker self-ligation products generated during RNA sequencing library construction, or a method of constructing an RNA sequencing library, comprising the steps of:

(1) Attaching the 3 'end linker to the 3' end of the sample RNA molecule;

(2) Ligating the 5' end linker to the 5' end of the sample RNA molecule ligated to the 3' end linker;

(3) Obtaining an extension product cDNA using an extension enzyme based on the sample RNA molecule to which the 3 '-end linker and the 5' -end linker are ligated, using an extension primer;

(4) Synthesizing a double-stranded synthesis product comprising a complementary strand of cDNA based on the extension product cDNA using a double-stranded synthesis primer; and

(5) Performing enzyme digestion treatment on the double-stranded synthesis product by using endonuclease to remove the self-ligation product;

wherein the 5 'end linker and the 3' end linker each comprise a partial sequence of a recognition site for the endonuclease, and a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker when the self-ligation product is formed;

optionally, PCR amplification is performed on the product obtained in step (4) or (5), and/or PCT amplification is included in step (5),

optionally, the amount of RNA molecules in the sample is more than or equal to 50pg; preferably, the amount of RNA molecules of said sample is between 50pg and 20ng.

3. The method of item 2, wherein the sample RNA molecules are small RNA molecules; preferably, the small RNA molecules are 15-200nt in length.

4. The method according to item 2 or 3, wherein the ligation reaction of step (1) uses truncated T4RNA ligase 2 as the ligase.

5. The method according to any one of items 1 to 4, wherein the 3 'end linker and the 5' end linker carry respectively more than 30%, preferably more than 40%, more preferably 50% of the sequence of the recognition site of the endonuclease, and a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker when the self-ligation product is formed.

6. The method according to any one of claims 2 to 5, wherein the elongation enzyme used in step (3) is reverse transcriptase or Bst polymerase.

7. The method according to any one of items 1 to 6, wherein the endonuclease is an endonuclease; preferably, the endonuclease is a double-stranded endonuclease; preferably, the endonuclease is selected from the group consisting of: <xnotran> AatII, bamHI, bsaBI, bsrFI, draI, hphI, ndeI, pvuII, swaI, acc65I, banI, bsaHI, bsrGI, draIII, hpy188I, ngoMI, rsaI, taqI, accI, banII, bsaI, bsrI, drdI, hpy188III, nheI, rsrII, tfiI, aciI, bbsI, bsaJi, bssHI, bssHII, eaeI, hpy99I, nlaIII, sacI, tliI, aclI, bbvCI, bsaWI, bssKI, eagI, hpyCH4III, nlaIV, sacII, tseI, acuI, bbvI, bsaXI, bssSI, earI, hpyCH4IV, notI, salI, tsp45I, afeI, bccI, bseRI, bstAPI, eciI, hpyCH4V, nruI, sapI, tsp509I, aflII, bceAI, bseYI, bstBI, ecoNI, kasI, nsiI, sau3AI, tspRI, aflIII, bcgI, bsgI, bstEII, ecoO109I, kpnI, nspI, sau96I, tth111I, ageI, bciVI, bsiEI, bstF5I, ecoRI, mboI, pacI, sbfI, xbaI, ahdI, bclI, bsiHKAI, bstNI, ecoRV, mboII, paeR7I, scaI, xcmI, aleI, bfaI, bsiWI, bstUI, fatI, mfeI, pciI, scrFI, xhoI, aluI, bfrBI, bsiI, bstXI, fauI, mluI, pflFI, sexAI, xmaI, alwI, bfuAI, bsmAI, bstYI, fnu4HI, mlyI, pflMI, sfaNI, xmnI, alwNI, bfuCI, bsmBI, bstZ17I, fseI, mmeI, phoI, sfcI, zraI, apaI, bglI, bsmFI, bsu36I, fspI, mnlI, pleI, sfoI, apaLI, bglII, bsmI, btgI, haeII, mscI, pmeI, sgrAI, nb.BbvCI, apeKI, blpI, bsoBI, btgZI, haeIII, mseI, pmlI, smaI, nt.BbvCI, apoI, bme1580I, bsp1286I, btsI, hgaI, msiI, ppuMI, smlI, nb.BsmI, ascI, bmgBI, bspCNI, cac8I, hhaI, mspA1I, pshAI, snaBI, nt.BstNBI, aseI, bmrI, bspDI, claI, hincII, mspI, psiI, speI, asiSI, bmtI, bspEI, cspCI, hindIII, mwoI, pspGI, sphI, avaI, bpmI, bspHI, cviAII, hinfI, naeI, pspOMI, sspI, avaII, bpu10I, bspMI, ddeI, hinP1I, narI, pspXI, stuI, avrII, bpuEI, bsrBI, dpnI, hpaI, nciI, pstI, styD4I, baeI, bsaAI, bsrDI, dpnII, hpaII, ncoI, pvuI, StyI, , DNA , RNA 50 , , DNA AsiSI, pacI, </xnotran> NotI, or BssHII.

8. A kit for constructing an RNA sequencing library, comprising:

an RNA3 'end ligation module comprising a3' end linker and for ligating the 3 'end linker to the 3' end of RNA;

an RNA5 'end ligation module comprising a5' end linker and for ligating the 5 'end linker to the 5' end of an RNA;

an extension module comprising an extension enzyme and for extending the RNA molecule to which the 3 'end linker and 5' end linker are attached;

a duplex synthesis module comprising a DNA polymerase and for synthesizing a duplex based on the product of the extension module;

an enzymatic cleavage module comprising an endonuclease and configured to remove adaptor self-ligated products within the products of the duplex synthesis module;

an amplification module which comprises an enzyme required by DNA amplification and is used for amplifying the product of the enzyme digestion module to obtain the sequencing library,

optionally, the RNA sequencing library is a small RNA molecule sequencing library; preferably, the small RNA molecule is 15-200nt in length.

9. The kit of claim 8, wherein the RNA3' end ligation module comprises a ligase, preferably the ligase is a truncated T4RNA ligase 2 or a point mutant thereof; and/or the RNA5' end ligation module comprises a ligase, preferably the ligase is T4RNA ligase 1.

10. The kit according to claim 8 or 9, wherein the elongase is AMV reverse transcriptase.

11. The kit according to any one of claims 8 to 10, wherein the endonuclease is an endonuclease; preferably, the endonuclease is a double-stranded endonuclease; preferably, the endonuclease is selected from the group consisting of: <xnotran> AatII, bamHI, bsaBI, bsrFI, draI, hphI, ndeI, pvuII, swaI, acc65I, banI, bsaHI, bsrGI, draIII, hpy188I, ngoMI, rsaI, taqI, accI, banII, bsaI, bsrI, drdI, hpy188III, nheI, rsrII, tfiI, aciI, bbsI, bsaJi, bssHI, bssHII, eaeI, hpy99I, nlaIII, sacI, tliI, aclI, bbvCI, bsaWI, bssKI, eagI, hpyCH4III, nlaIV, sacII, tseI, acuI, bbvI, bsaXI, bssSI, earI, hpyCH4IV, notI, salI, tsp45I, afeI, bccI, bseRI, bstAPI, eciI, hpyCH4V, nruI, sapI, tsp509I, aflII, bceAI, bseYI, bstBI, ecoNI, kasI, nsiI, sau3AI, tspRI, aflIII, bcgI, bsgI, bstEII, ecoO109I, kpnI, nspI, sau96I, tth111I, ageI, bciVI, bsiEI, bstF5I, ecoRI, mboI, pacI, sbfI, xbaI, ahdI, bclI, bsiHKAI, bstNI, ecoRV, mboII, paeR7I, scaI, xcmI, aleI, bfaI, bsiWI, bstUI, fatI, mfeI, pciI, scrFI, xhoI, aluI, bfrBI, bsiI, bstXI, fauI, mluI, pflFI, sexAI, xmaI, alwI, bfuAI, bsmAI, bstYI, fnu4HI, mlyI, pflMI, sfaNI, xmnI, alwNI, bfuCI, bsmBI, bstZ17I, fseI, mmeI, phoI, sfcI, zraI, apaI, bglI, bsmFI, bsu36I, fspI, mnlI, pleI, sfoI, apaLI, bglII, bsmI, btgI, haeII, mscI, pmeI, sgrAI, nb.BbvCI, apeKI, blpI, bsoBI, btgZI, haeIII, mseI, pmlI, smaI, nt.BbvCI, apoI, bme1580I, bsp1286I, btsI, hgaI, msiI, ppuMI, smlI, nb.BsmI, ascI, bmgBI, bspCNI, cac8I, hhaI, mspA1I, pshAI, snaBI, nt.BstNBI, aseI, bmrI, bspDI, claI, hincII, mspI, psiI, speI, asiSI, bmtI, bspEI, cspCI, hindIII, mwoI, pspGI, sphI, avaI, bpmI, bspHI, cviAII, hinfI, naeI, pspOMI, sspI, avaII, bpu10I, bspMI, ddeI, hinP1I, narI, pspXI, stuI, avrII, bpuEI, bsrBI, dpnI, hpaI, nciI, pstI, styD4I, baeI, bsaAI, bsrDI, dpnII, hpaII, ncoI, pvuI, StyI, </xnotran>

More preferably, the endonuclease is bioinformatically screened, more preferably the endonuclease is present in a number of less than 50 in a small RNA sequence database, and even more preferably the endonuclease is AsiSI, pacI, notI, or bshii.

12. Use of the method according to any one of claims 1-7 or the kit according to any one of claims 8-11 for constructing an RNA sequencing library.

Preferably, the RNA sequencing library is selected from the group consisting of: plasma small RNA sequencing library, CLIP library, RIP library, meRIP library and GRO library etc.

13. A method of determining sequence information of a small RNA molecule, comprising:

constructing a sequencing library based on the sample of small RNA molecules by the method according to item 2; sequencing the sequencing library to obtain a sequencing result; and obtaining sequence information of the small RNA molecule based on the sequencing result.

14. A system for determining sequence information of a small RNA molecule, comprising:

the kit according to any one of claims 8 to 11;

the sequencing device is used for sequencing the sequencing library constructed on the sample by the kit to obtain a sequencing result of the sample; and

and the analysis device analyzes the sequencing result to obtain the sequence information of the small RNA molecule.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic flow diagram of a method for removing adaptor self-ligation products during sequencing library construction and a small RNA library construction method according to one embodiment of the invention;

FIG. 2 shows a fragment size quality control plot of a small RNA library constructed according to one embodiment of the present invention. Wherein the self-connection length of the joint is 141nt, and the input amount is 1ng of miRNA sample.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"endonuclease" refers to an enzyme that can hydrolyze phosphodiester bonds in the interior of a nucleic acid molecule chain to produce an oligonucleotide, and can be classified into a DNA endonuclease that decomposes DNA and an RNA endonuclease that decomposes RNA. Endonucleases include "DNA restriction endonucleases", which are also referred to simply as restriction endonucleases or restriction enzymes, refer to a class of active proteins that recognize a specific nucleotide sequence and cleave the phosphodiester bond between two nucleotides at a specific site in each nucleic acid strand. The distribution of restriction endonucleases is extremely wide, and at least one restriction endonuclease is found in almost all genera and species of bacteria. Common endonucleases include, but are not limited to, those listed in the following table:

table I common endonucleases

Each of which recognizes a specific sequence and cuts the DNA sequence within this sequence in a specific manner. This particular sequence that the endonuclease recognizes and cuts is also referred to herein as a "recognition site" or "cut site". As long as a recognition site for a certain endonuclease exists in a DNA sequence, the DNA sequence can be cleaved with the enzyme to perform various molecular biological operations.

"Gene sequencing" refers to the analysis of the arrangement of base sequences in DNA or RNA fragments by a certain technical analysis means, thereby providing support for biological and medical research findings.

"sequencing library" refers to a collection of random fragments of all DNA or RNA in a species or sample used for gene sequencing.

DNA (i.e., deoxyribonucleic acid) refers to a nucleic acid within a biological cell that carries the genetic information necessary for the synthesis of RNA and proteins, and is a biological macromolecule essential for the development and proper functioning of an organism. DNA is a macromolecular polymer composed of deoxyribonucleotides. Deoxyribonucleotides are composed of a base, deoxyribose, and phosphate. There are 4 bases, adenine (A), guanine (G), cytosine (C) and thymine (T). RNA (i.e., ribonucleic acid) refers to a genetic information carrier within a biological cell, a partial virus, or a viroid. RNA is a macromolecular polymer composed of ribonucleotides. Ribonucleotides are composed of a base, a ribose, and a phosphate. There are 4 bases, adenine (A), guanine (G), cytosine (C) and uracil (U).

The 5 '-end of DNA or RNA refers to the end of a single strand of DNA or RNA that carries a free 5' -hydroxyl group or a phosphate thereof. 3 'end of DNA or RNA the single strand of DNA or RNA has a free 3' -hydroxyl group or a phosphate terminus thereof. cDNA in the present invention refers to a DNA strand complementary to RNA after reverse transcription in vitro.

Small RNA means in the present invention RNA molecules of 15 to 300nt, preferably 15 to 200nt, more preferably 20 to 200nt in length, which comprise: (1) Micrornas (i.e., mirnas), which are small RNAs of about 20-24 nucleotides in length that are endogenous, are generated by Dicer enzyme processing of single-stranded RNA precursors of about 70-90 bases in size that are hairpin structures. It has a number of important regulatory roles within the cell. Each miRNA can have multiple target genes, and several miRNAs can also regulate the same gene; (2) Small interfering RNAs (i.e., siRNAs, sometimes also referred to as short interfering RNAs or silencing RNAs), which refer to a class of double-stranded RNAs of about 20-25 nucleotides in length, are primarily involved in RNA interference phenomena and regulate gene expression in a specific manner; (3) Piwi-interacting RNA (i.e., piRNA), which refers to a class of small RNA molecules that are about 29-30 nucleotides in length. It is mainly present in germ cells and stem cells of mammals and regulates gene silencing pathways by binding to Piwi subfamily proteins to form piRNA complexes (pircs); (4) Small nuclear RNA (i.e., snRNA), which is the major component of RNA spliceosomes during post-transcriptional processing in eukaryotes, involved in the processing of mRNA precursors; (5) Nucleolar small RNA (i.e., snoRNA), which refers to small non-coding RNA encoded by introns and distributed throughout the nucleoli of eukaryotic cells, has conserved structural elements. Antisense snoRNA has been shown to direct rRNA ribomethylation; (6) Small ribosomal RNA (i.e., srRNA), which refers to small ribosomal RNA that is predominantly distributed in the rDNA coding region and matches the sense strand. Srna has been found to bind specifically to AGO proteins and its expression profile is correlated with diabetes.

Small RNA samples are obtained that can be used to build RNA sequencing libraries. The small RNA of the invention can be quantified by a Qubit microRNA assay kit and a Qubit 4.0 fluorescence quantifier. The amount of the small RNA sample of the invention can be more than or equal to 50pg, and preferably 50pg-20ng.

Linking the small RNA sample to the 3' end linker refers to linking the 3' end linker to the 3' end of the small RNA sample molecule. The 3 'end linker of the invention refers to any short nucleic acid sequence capable of linking to the 3' end of an RNA molecule. In one embodiment, the linker is a short nucleic acid sequence linked to the 3' end of the RNA molecule by a truncated form of T4RNA ligase 2 or a point mutant thereof. The enzyme is only capable of attaching an adenylated single-stranded DNA or RNA linker to the 3' end of an RNA molecule, thereby avoiding self-ligation between small RNA molecules. In one embodiment, the linker has an adenylation modification at the 5 'end and a dideoxy modification at the 3' end, thereby preventing self-ligation of the linker in the ligation reaction. In one embodiment, the 5' end of the linker carries a partial sequence of the recognition site of the endonuclease of the present invention, preferably a sequence of at least 30%, preferably at least 40%, more preferably 50% (half) of the recognition site. The sequence of the linker may be, for example, 5'-rAPP-CGCAAGTCGGAGGCCAAG-3' ddC. In one embodiment, the linking may be performed in a reaction system comprising: small RNA molecule sample, 3' end joint, truncated T4RNA ligase 2. In one embodiment, the reaction system further comprises a ligase reaction buffer, PEG8000 and an rnase inhibitor. In one embodiment, the small RNA molecule sample is subjected to a heat denaturation treatment (e.g., a reaction at 70 ℃ for 2 minutes). In one embodiment, the linking may be performed under the following reaction conditions: at 16 ℃ overnight or at 25 ℃ for 2 hours or at 37 ℃ for 30 minutes.

Linking the small RNA molecule to which the 3' end linker is linked to the 5' end linker means linking the 5' end linker to the 5' end of the small RNA sample molecule to which the 3' end linker is linked. The 5' end linker of the present invention refers to any short nucleic acid sequence capable of linking to the 5' end of a small RNA sample molecule, preferably a short nucleic acid sequence linked to the 5' end of an RNA molecule by T4RNA ligase 1. In one embodiment, the 3' end of the linker carries a partial sequence of the recognition site for the endonuclease of the present invention, preferably at least 30%, preferably at least 40%, more preferably 50% (the other half) of the sequence of the recognition site. The sequence of the linker may be, for example, 5'-GCUACGAUCCGACUUNNNNGCG-3'. In one embodiment, the linking may be performed in a reaction system comprising: a small RNA sample molecule with a3 'end linker attached, a5' end linker, T4RNA ligase 1. In one embodiment, the reaction system further comprises a ligase reaction buffer and PEG8000. In one embodiment, the linking may be performed under the following reaction conditions: the reaction was carried out at 16 ℃ overnight or at 25 ℃ for 2 hours.

Reverse transcription refers to a process of synthesizing DNA using RNA as a template, i.e., DNA synthesis under RNA guidance. The direction of nucleic acid synthesis and the direction of flow of genetic information (RNA to DNA) in this process are both opposite to the transcription process (DNA to RNA) and are therefore termed reverse transcription. Reverse transcription is performed by extending in the 5' → 3' direction on the 3' -end of a reverse transcription primer using dNTP as a substrate and RNA as a template with the aid of a reverse transcriptase to synthesize a cDNA single strand complementary to the RNA template, which forms an RNA-cDNA hybrid with the RNA template. Then, the RNA strand is hydrolyzed under the action of reverse transcriptase to obtain cDNA. The reverse transcription primer may be a random primer or a gene-specific primer. In one embodiment, reverse transcription is referred to as an extension reaction and the reverse transcription primer is referred to as an extension primer, which is a sequence designed according to the known sequence of the linker attached at the 3' end of the RNA molecule of the present invention, which may be, for example, 5-. In one embodiment, the extension may be performed in a reaction system comprising: small RNA sample molecules linked to a3 'end linker and a5' end linker, extension primers and reverse transcriptase. In one embodiment, the reaction system further comprises an extension reaction buffer. In one embodiment, the linking may be performed under the following reaction conditions: the reaction was carried out at 42 ℃ for 30 minutes.

Double-strand synthesis refers to a process of synthesizing a second DNA strand using cDNA obtained by reverse transcription as a template. Wherein a double-stranded synthesis product comprising the complementary strand of cDNA is synthesized based on the extension product cDNA obtained by the extension reaction of the present invention using a double-stranded synthesis primer. The two-strand synthesis primer is a sequence designed based on the known sequence of cDNA obtained by the extension reaction of the present invention, and may be, for example, 5-. In one embodiment, the two-chain synthesis may be carried out in a reaction system comprising: extension products, duplex synthesis primers and DNA polymerase. In one embodiment, the reaction system further comprises a DNA polymerase buffer. In one embodiment, the linking may be performed under the following reaction conditions: 2 minutes at 98 ℃,30 seconds at 60 ℃ and 5 minutes at 72 ℃.

The enzyme digestion treatment refers to a process of using an endonuclease to cleave a sequence self-ligated to a linker at a recognition site of an intact enzyme formed at the junction of a5 'end linker and a3' end linker. The reaction conditions for the cleavage vary depending on the DNA endonuclease used, and are usually 20 to 60 ℃ and preferably 28 to 50 ℃. In one embodiment, the endonuclease used is BssHII, and the cleavage conditions used are incubation of the sequence to be cleaved with the enzyme at 50 ℃ for 30 minutes.

The second generation high throughput sequencing is a technology of sequencing while synthesizing by using microbeads or high density chips, and has the advantages of high sequencing throughput and capability of obtaining data G at one time.

The amplification or PCR amplification in the present invention means: a target DNA double strand containing a sequence to be amplified and analyzed is treated at a high temperature for a period of time to form two oligonucleotide single strands (unzipped), and a pair of oligonucleotide fragments which are artificially synthesized according to a known DNA sequence and are complementary to adjacent sequences at both ends of the amplified DNA are added as amplification primers, namely, forward and reverse primers. The primer contacts with two DNA single strands at a lower temperature for a period of time to complementarily bind (anneal) respectively, and extends for a period of time in the 5 'to 3' direction at a higher temperature and under the action of DNA polymerase with the target DNA single strand as a template and 4 kinds of mononucleotides (dNTPs) as raw materials, thereby synthesizing a new DNA double strand (extension). Then, another melting-annealing-extension cycle is started again, so that a very small amount of the target DNA is specifically amplified by a million-fold or more (i.e., an amplification product). In one embodiment, the amplification of the present invention is to synthesize a PCR reaction product based on the product obtained after the enzymatic cleavage of the present invention using PCR primers. PCR primers are sequences designed based on the known sequences of the extension primers and the two-strand synthesis primers of the present invention, and in one embodiment, a certain number of random bases may be present in the forward and reverse primers for use in differentiating different samples in high-throughput sequencing. The forward and reverse primers can be respectively as follows: for example, the forward primer 5 '-GCATGGCGACCTTACGNNNNNNNNNNNNTGTCTTCTCTCAAAGACCGCTTGG-3' (where the sequence indicated in bold is the 5 'sequence of the extension primer) and the reverse primer 5' -Pho-CTCTCAGTACGTCAGCAGTTNNNNNNNNNNNNCAACTCCTTGCTCACAGAAC-3 '(where the sequence indicated in bold is the 5' sequence of the two-strand synthesis primer) where 10 random bases are used to distinguish different samples in high throughput sequencing. In one embodiment, the PCR amplification may be performed in a reaction system comprising: enzyme products, forward primer, reverse primer and DNA polymerase. In one embodiment, the DNA polymerase is a high fidelity DNA polymerase. In one embodiment, the reaction system further comprises a DNA polymerase buffer and dNTPs. In one embodiment, the linking may be performed under the following reaction conditions: 2 minutes at 98 ℃; followed by 15-30, preferably 15-20, more preferably 15-18 cycles of 98 ℃ for 15 seconds, 60 ℃ for 30 seconds and 72 ℃ for 30 seconds; then reacted at 72 ℃ for 5 minutes. In one embodiment, the PCR product is purified by the magnetic bead method.

In order to solve the technical problems in the prior art, the present invention aims to provide a method for removing a linker self-ligation product in a sequencing library construction process. The method can specifically cut the adaptor self-ligation product by utilizing the substrate selectivity of the DNA endonuclease, thereby realizing the simple and efficient library construction of the small RNA or RNA fragments of a trace sample.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

according to one aspect of the present invention, there is provided a method for cleaving a linker self-ligation product using an endonuclease, the method comprising: mixing the endonuclease and the linker self-ligation product, wherein the linker self-ligation product comprises a recognition site for the endonuclease; the endonuclease can cleave at the recognition site.

In one embodiment, the adaptor self-ligation product is generated during the construction of a sequencing library. In one embodiment, the adaptor self-ligation product is formed by ligation of a5 'end adaptor and a3' end adaptor used in the sequencing library construction process. In one embodiment, the 5 'end linker and the 3' end linker each comprise a partial sequence of the recognition site, and upon formation of the linker self-ligation product, a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker. In one embodiment, the sequencing library is an RNA sequencing library. In one embodiment, the 3 'end linker and the 5' end linker each carry more than 30%, preferably more than 40%, more preferably 50% of the sequence of the recognition site of the endonuclease, and the complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker when the self-ligation product is formed. In one embodiment, the endonuclease is an endonuclease. In one embodiment, the endonuclease may be selected from the group of endonucleases listed in table I.

According to another aspect of the present invention, there is provided a method of removing 5 'and 3' adaptor self-ligation products generated during the construction of an RNA sequencing library, or a method of constructing an RNA sequencing library, comprising the steps of:

(1) Attaching a3 'end linker to the 3' end of the RNA molecule;

(2) Connecting a5' end connector to the 5' end of the RNA molecule connected with the 3' end connector;

(3) Obtaining an extension product using an extension enzyme based on the RNA molecule to which the 3 'end linker and the 5' end linker are ligated, using an extension primer;

(4) Synthesizing a double-stranded synthesis product based on the extension product using a double-stranded synthesis primer;

(5) And (3) carrying out enzyme digestion treatment on the double-strand synthesis product by using endonuclease to remove the adaptor self-connection product in the system.

In one embodiment, the 3 'end linker and the 5' end linker each comprise a partial sequence of the recognition site of the endonuclease, for example more than 30%, preferably more than 40%, more preferably 50% of the sequence of the recognition site, and the complete sequence of the recognition site is formed at the junction of the 5 'end linker and the 3' end linker when the linker self-ligation product is formed. In one embodiment, PCR amplification is performed based on the sequence obtained in step (5), and the obtained amplification product is the RNA sequencing library. In one embodiment, the amount of the RNA molecule is ≧ 50pg. In one embodiment, the amount of said RNA molecule is between 50pg and 20ng. In one embodiment, the RNA molecule is a small RNA molecule. In one embodiment, the small RNA molecule is 15-200nt in length. In one embodiment, the ligation reaction of step (1) uses truncated T4RNA ligase 2 as the ligase. In one embodiment, the extension enzyme used in step (3) is reverse transcriptase or Bst polymerase. In one embodiment, the endonuclease is an endonuclease. In one embodiment, the endonuclease may be selected from the group of endonucleases listed in table I.

By using the method for removing the 5 'and 3' adaptor self-connection byproducts generated in the construction of the sequencing library, the sequencing library of the small RNA molecules in a trace sample can be effectively constructed, and particularly the small RNA sequencing library of the trace sample such as a plasma sample and a single-cell sample can be effectively constructed. In particular, the library building process provided by the invention can select a tubular reaction process, thereby further reducing the investment of building the library and reducing the deviation of the sequencing result caused by the purification process.

According to another aspect of the invention, a kit for constructing a small RNA sequencing library is provided. According to an embodiment of the invention, the kit comprises: an RNA3 'end ligation module comprising a3' end linker and for ligating the 3 'end linker to the 3' end of the RNA; an RNA5' end ligation module comprising a5' end linker and for ligating a5' end linker to the 5' end of the RNA to which the 3' end linker is ligated; an extension module comprising an extension enzyme and for extending the RNA molecule to which the 3 'end linker and 5' end linker are attached; a duplex synthesis module comprising a DNA polymerase and for synthesizing a duplex based on the product of the extension module; an enzymatic cleavage module comprising an endonuclease and configured to remove linker self-ligation products from the products of the duplex synthesis module. And the amplification module contains enzymes required by DNA amplification and is used for amplifying enzyme digestion products to obtain a final library.

In one embodiment, the RNA3' end ligation module comprises a ligase, preferably the ligase is a truncated T4RNA ligase 2 or a point mutant thereof. In one embodiment, the RNA5' end ligation module comprises a ligase, preferably the ligase is T4RNA ligase 1. In one embodiment, the elongase is an AMV reverse transcriptase. In one embodiment, the endonuclease is an endonuclease. In one embodiment, the endonuclease may be selected from the group of endonucleases listed in table I. In one embodiment, the endonuclease is bioinformatically screened. In one embodiment, the recognition sites for the endonuclease are present in a number of less than 50 in a small RNA sequence database. In one embodiment, the endonuclease is AsiSI, pacI, notI, or bshii.

By utilizing the kit for constructing the sequencing library of the small RNA molecules, the construction of the sequencing library can be carried out by utilizing a trace small RNA sample, the sample loss is less, the sequence information is kept complete, and the kit can be applied to the library construction of small RNA samples extracted from plasma and small RNA samples in single cells.

The RNA sample may be obtained from any source, including but not limited to organisms, organs, tissues, cells, organelles, and the like.

According to a further aspect of the invention there is provided the use of a method according to the invention or a kit according to the invention in the construction of an RNA sequencing library. In one embodiment, the RNA sequencing library includes, but is not limited to: plasma small RNA sequencing library, CLIP library, RIP library, meRIP library and GRO library etc.

According to yet another aspect of the invention, there is provided a method of determining sequence information of a small RNA molecule, according to an embodiment of the invention, the method comprising: constructing a sequencing library according to the method of the invention based on a small RNA sample; sequencing the sequencing library to obtain a sequencing result; and determining sequence information of the small RNA molecule based on the sequencing result.

According to yet another aspect of the invention, a system for determining sequence information of a small RNA molecule is provided. According to an embodiment of the invention, the system comprises: the kit comprises a sequencing library constructing device, a sequencing library constructing device and a sequencing library constructing device, wherein the sequencing library constructing device is the kit; the sequencing device is used for sequencing the sequencing library constructed by the kit on the sample to obtain a sequencing result of the sample; and an analysis device for analyzing the sequencing result of the sample so as to obtain the sequence information of the small RNA molecule.

By adopting the system for determining the sequence information of the small RNA molecules according to the embodiment of the invention, the sequence information of the small RNA in a trace sample can be determined sensitively, accurately and efficiently.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Examples

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. It will be appreciated by those skilled in the art that the embodiments described below with reference to the accompanying drawings are illustrative only and are not to be construed as limiting the invention. The examples, where specific technical conditions are not indicated, are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. The reagents or instruments used are conventional products available from commercial markets, such as NEB and others, without reference to the manufacturer.

According to one aspect of the invention, the invention provides a method for removing adaptor self-ligation during the construction of an RNA library and a method for constructing a small RNA library. Referring to fig. 1, according to an embodiment of the invention, the method may include the steps of:

1. the small RNA samples used for pooling were first quantified with a Qubit 4.0 and quantified using a Qubit microRNA assay kit. For the small RNA fraction of the pool, 50pg-20ng can be used as the starting amount.

2. The small RNA molecules are ligated to a3' end linker.

2-1 mixing the small RNA molecule sample with the 3' end linker, reacting at 70 ℃ for 2 minutes, and then performing ligation in a ligation reaction system using ligase.

The sequence of the 2-2 ' end linker is 5' -rAPP-CGCAAGTCGGAGGCCAAG-3' ddC, the 5' end of the linker sequence has an adenylation modification, and the 3' end has a dideoxy modification, preventing self-ligation of the linker in the ligation reaction. And a half recognition sequence of BssHII enzyme is arranged at the 5' end of the joint for the subsequent joint self-ligation removal process.

2-3 the ligase for this ligation reaction is a truncated T4RNA ligase 2 and its various point mutants, which are only capable of ligating an adenylated single-stranded DNA or RNA linker to the 3' end of an RNA molecule, thereby avoiding self-ligation between small RNA molecules.

The 2-4 connection reaction system comprises: 2-1, a3' end connector, a truncated T4RNA ligase 2, a ligase reaction buffer, PEG8000 and an RNase inhibitor.

The reaction conditions of 2-5 are 16 ℃ overnight or 25 ℃ for 2 hours or 37 ℃ for 30 minutes.

3. The small RNA molecules with the 3 'end linker attached are ligated to the 5' end linker.

3-1 the sample of small RNA molecules to which the 3 '-end linker is ligated is mixed with the 5' -end linker, reacted at 70 ℃ for 2 minutes, and then ligated in a ligation reaction system using ligase.

The 3-2 'linker sequence is 5' -GCUACGAUCCGACUUNNNNGCG-3', and the 3' end of the linker sequence has the other half of the recognition sequence of BssHII enzyme for the subsequent self-ligation removal process of the linker.

3-3 the ligation reaction system comprises: 2-5, a5' end linker, PEG8000, ligase buffer and T4RNA ligase 1.

3-4 the reaction conditions were 16 ℃ overnight or 25 ℃ for 2 hours.

4. And (3) carrying out extension reaction.

4-1 extension is carried out using the ligation product obtained in 3-4 as a template and an extension primer designed based on the known sequence of the 3' end linker to obtain an extension product.

The sequence of the 4-2 extension primer is as follows: 5' TTGTCTTCCTAAGACCGCTCCTTGGCCTCCGACTTGCG-3

The 4-3 extension reaction system comprises: 3-4, extension primer, extension reaction buffer solution and reverse transcriptase.

4-4 reaction conditions: the reaction was carried out at 42 ℃ for 30 minutes.

4-5 hydrolyzing RNA chain under the action of reverse transcriptase to obtain extension product cDNA.

5. And (3) performing a two-chain synthesis reaction.

5-1 Using the extension product obtained in 4-5 as a template, a double-strand synthesis product containing the complementary strand of cDNA was synthesized using a double-strand synthesis primer designed based on the known sequence of cDNA obtained in 4-5.

The 5-2 two-strand synthesis primer sequence is as follows: 5' CAACTCCTTGGCTCACAGAACGACATGGCATACGTCACGATCCGACTT-3

5-3 the reaction system comprises: 4-5, a primer for double-strand synthesis, a DNA polymerase buffer solution and a DNA polymerase

5-4 reaction conditions are as follows: : denaturation at 98 ℃ for 2 min; 30 seconds at 60 ℃; the reaction was carried out at 72 ℃ for 5 minutes.

6. And (4) carrying out enzyme digestion treatment.

6-1, carrying out enzyme digestion treatment on the two-chain synthetic product obtained from 5-4, and removing the self-connection of the joint.

6-2 the endonuclease used was BssHII.

6-3 the reaction conditions were 50 ℃ for 30 minutes.

And 7.PCR amplification.

7-1, taking the enzyme digestion product obtained in the step 6-3 as a template, and carrying out PCR amplification by utilizing forward and reverse primers designed according to the known sequences of an extension primer and a double-strand synthesis primer, wherein a certain number of random basic groups can be introduced into the forward and reverse primers to be used for distinguishing different samples in high-throughput sequencing.

7-2 Synthesis of Forward and reverse PCR primers

Forward primer sequence:

5’-GCATGGCGACCTTATCAGNNNNNNNNNNTTGTCTTCCTAAGACCGCTTGG-3’

reverse primer sequence:

5’-Pho-CTCTCAGTACGTCAGCAGTTNNNNNNNNNNCAACTCCTTGGCTCACAGAAC-3’

10 of these random bases were used to distinguish different samples in high throughput sequencing.

The 7-3 reaction system comprises: 6-3, forward and reverse PCR primers, high-fidelity DNA polymerase and DNA polymerase buffer solution.

7-4 reaction conditions: denaturation at 98 ℃ for 2 min; 98 deg.C, 15 seconds, 60 deg.C, 30 seconds, 72 deg.C, 30 seconds. The process is cyclically reacted for 15-18 cycles. Then reacted at 72 ℃ for 5 minutes.

8. And (4) purifying the PCR product by a magnetic bead method to obtain a target product, and recovering to obtain a library.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. Those skilled in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (14)

1. A method for cleaving a linker self-ligation product using an endonuclease, the method comprising mixing the endonuclease and the linker self-ligation product, wherein the linker self-ligation product comprises a recognition site for the endonuclease at which the endonuclease cleaves;

optionally, the adaptor self-ligation product is generated during construction of a sequencing library;

optionally, the adaptor self-ligation product is formed by ligation of a5 'end adaptor and a3' end adaptor used in the sequencing library construction process;

optionally, the 5 'end linker and the 3' end linker each comprise a partial sequence of the recognition site, and upon formation of the linker self-ligation product, a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker;

optionally, the sequencing library is an RNA sequencing library.

2. A method of removing 5 'end linker and 3' end linker self-ligation products generated during RNA sequencing library construction, or a method of constructing an RNA sequencing library, comprising the steps of:

(1) Attaching the 3 'end linker to the 3' end of the sample RNA molecule;

(2) Ligating a5' end linker to the 5' end of the sample RNA molecule to which the 3' end linker is ligated;

(3) Obtaining an extension product cDNA using an extension enzyme based on the sample RNA molecule to which the 3 '-end linker and the 5' -end linker are ligated, using an extension primer;

(4) Synthesizing a double-stranded synthesis product comprising a complementary strand of cDNA based on the extension product cDNA using a double-stranded synthesis primer; and

(5) Performing enzyme digestion treatment on the double-stranded synthesis product by using endonuclease to remove the self-ligation product;

wherein the 5 'end linker and the 3' end linker each comprise a partial sequence of a recognition site for the endonuclease, and a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker when the self-ligation product is formed;

optionally, PCR amplification is performed on the product obtained in step (4) or (5), and/or PCT amplification is included in step (5),

optionally, the amount of RNA molecules in the sample is more than or equal to 50pg; preferably, the amount of RNA molecules of said sample is between 50pg and 20ng.

3. The method of claim 2, wherein the sample RNA molecules are small RNA molecules; preferably, the small RNA molecules are 15-200nt in length.

4. The method according to claim 2 or 3, wherein the ligation reaction of step (1) uses truncated T4RNA ligase 2 as ligase.

5. The method according to any one of claims 1-4, wherein the 3 'end linker and the 5' end linker carry a sequence of more than 30%, preferably more than 40%, more preferably 50% of the recognition site sequence of the endonuclease, respectively, and a complete recognition site sequence is formed at the junction of the 5 'end linker and the 3' end linker when the self-ligation product is formed.

6. The method according to any one of claims 2 to 5, wherein the elongase used in step (3) is reverse transcriptase or Bst polymerase.

7. The method according to any one of claims 1-6, wherein the endonuclease is an endonuclease; preferably, the endonuclease is a double-stranded endonuclease; preferably, the endonuclease is selected from the group consisting of: <xnotran> AatII, bamHI, bsaBI, bsrFI, draI, hphI, ndeI, pvuII, swaI, acc65I, banI, bsaHI, bsrGI, draIII, hpy188I, ngoMI, rsaI, taqI, accI, banII, bsaI, bsrI, drdI, hpy188III, nheI, rsrII, tfiI, aciI, bbsI, bsaJi, bssHI, bssHII, eaeI, hpy99I, nlaIII, sacI, tliI, aclI, bbvCI, bsaWI, bssKI, eagI, hpyCH4III, nlaIV, sacII, tseI, acuI, bbvI, bsaXI, bssSI, earI, hpyCH4IV, notI, salI, tsp45I, afeI, bccI, bseRI, bstAPI, eciI, hpyCH4V, nruI, sapI, tsp509I, aflII, bceAI, bseYI, bstBI, ecoNI, kasI, nsiI, sau3AI, tspRI, aflIII, bcgI, bsgI, bstEII, ecoO109I, kpnI, nspI, sau96I, tth111I, ageI, bciVI, bsiEI, bstF5I, ecoRI, mboI, pacI, sbfI, xbaI, ahdI, bclI, bsiHKAI, bstNI, ecoRV, mboII, paeR7I, scaI, xcmI, aleI, bfaI, bsiWI, bstUI, fatI, mfeI, pciI, scrFI, xhoI, aluI, bfrBI, bsiI, bstXI, fauI, mluI, pflFI, sexAI, xmaI, alwI, bfuAI, bsmAI, bstYI, fnu4HI, mlyI, pflMI, sfaNI, xmnI, alwNI, bfuCI, bsmBI, bstZ17I, fseI, mmeI, phoI, sfcI, zraI, apaI, bglI, bsmFI, bsu36I, fspI, mnlI, pleI, sfoI, apaLI, bglII, bsmI, btgI, haeII, mscI, pmeI, sgrAI, nb.BbvCI, apeKI, blpI, bsoBI, btgZI, haeIII, mseI, pmlI, smaI, nt.BbvCI, apoI, bme1580I, bsp1286I, btsI, hgaI, msiI, ppuMI, smlI, nb.BsmI, ascI, bmgBI, bspCNI, cac8I, hhaI, mspA1I, pshAI, snaBI, nt.BstNBI, aseI, bmrI, bspDI, claI, hincII, mspI, psiI, speI, asiSI, bmtI, bspEI, cspCI, hindIII, mwoI, pspGI, sphI, avaI, bpmI, bspHI, cviAII, hinfI, naeI, pspOMI, sspI, avaII, bpu10I, bspMI, ddeI, hinP1I, narI, pspXI, stuI, avrII, bpuEI, bsrBI, dpnI, hpaI, nciI, pstI, styD4I, baeI, bsaAI, bsrDI, dpnII, hpaII, ncoI, pvuI, StyI, , DNA , RNA 50 , , DNA AsiSI, pacI, </xnotran> NotI, or BssHII.

8. A kit for constructing an RNA sequencing library, comprising:

an RNA3 'end ligation module comprising a3' end linker and for ligating the 3 'end linker to the 3' end of RNA;

an RNA5 'end ligation module comprising a5' end linker and for ligating the 5 'end linker to the 5' end of RNA;

an extension module comprising an extension enzyme and for extending the RNA molecule to which the 3 'end linker and 5' end linker are attached;

a duplex synthesis module comprising a DNA polymerase and for synthesizing a duplex based on the product of the extension module;

an enzymatic cleavage module comprising an endonuclease and configured to remove adaptor self-ligated products within the products of the duplex synthesis module;

an amplification module which comprises enzymes required by DNA amplification and is used for amplifying products of the enzyme digestion module to obtain the sequencing library,

optionally, the RNA sequencing library is a small RNA molecule sequencing library; preferably, the small RNA molecules are 15-200nt in length.

9. The kit of claim 8, wherein the RNA3' end ligation module comprises a ligase, preferably the ligase is a truncated T4RNA ligase 2 or a point mutant thereof; and/or the RNA5' end ligation module comprises a ligase, preferably the ligase is T4RNA ligase 1.

10. The kit of claim 8 or 9, wherein the elongase is AMV reverse transcriptase.

11. The kit according to any one of claims 8-10, wherein the endonuclease is an endonuclease; preferably, the endonuclease is a double-stranded endonuclease; preferably, the endonuclease is selected from the group consisting of: <xnotran> AatII, bamHI, bsaBI, bsrFI, draI, hphI, ndeI, pvuII, swaI, acc65I, banI, bsaHI, bsrGI, draIII, hpy188I, ngoMI, rsaI, taqI, accI, banII, bsaI, bsrI, drdI, hpy188III, nheI, rsrII, tfiI, aciI, bbsI, bsaJi, bssHI, bssHII, eaeI, hpy99I, nlaIII, sacI, tliI, aclI, bbvCI, bsaWI, bssKI, eagI, hpyCH4III, nlaIV, sacII, tseI, acuI, bbvI, bsaXI, bssSI, earI, hpyCH4IV, notI, salI, tsp45I, afeI, bccI, bseRI, bstAPI, eciI, hpyCH4V, nruI, sapI, tsp509I, aflII, bceAI, bseYI, bstBI, ecoNI, kasI, nsiI, sau3AI, tspRI, aflIII, bcgI, bsgI, bstEII, ecoO109I, kpnI, nspI, sau96I, tth111I, ageI, bciVI, bsiEI, bstF5I, ecoRI, mboI, pacI, sbfI, xbaI, ahdI, bclI, bsiHKAI, bstNI, ecoRV, mboII, paeR7I, scaI, xcmI, aleI, bfaI, bsiWI, bstUI, fatI, mfeI, pciI, scrFI, xhoI, aluI, bfrBI, bsiI, bstXI, fauI, mluI, pflFI, sexAI, xmaI, alwI, bfuAI, bsmAI, bstYI, fnu4HI, mlyI, pflMI, sfaNI, xmnI, alwNI, bfuCI, bsmBI, bstZ17I, fseI, mmeI, phoI, sfcI, zraI, apaI, bglI, bsmFI, bsu36I, fspI, mnlI, pleI, sfoI, apaLI, bglII, bsmI, btgI, haeII, mscI, pmeI, sgrAI, nb.BbvCI, apeKI, blpI, bsoBI, btgZI, haeIII, mseI, pmlI, smaI, nt.BbvCI, apoI, bme1580I, bsp1286I, btsI, hgaI, msiI, ppuMI, smlI, nb.BsmI, ascI, bmgBI, bspCNI, cac8I, hhaI, mspA1I, pshAI, snaBI, nt.BstNBI, aseI, bmrI, bspDI, claI, hincII, mspI, psiI, speI, asiSI, bmtI, bspEI, cspCI, hindIII, mwoI, pspGI, sphI, avaI, bpmI, bspHI, cviAII, hinfI, naeI, pspOMI, sspI, avaII, bpu10I, bspMI, ddeI, hinP1I, narI, pspXI, stuI, avrII, bpuEI, bsrBI, dpnI, hpaI, nciI, pstI, styD4I, baeI, bsaAI, bsrDI, dpnII, hpaII, ncoI, pvuI, StyI, </xnotran>

More preferably, the endonuclease is bioinformatically screened, more preferably, the endonuclease is present in a number of less than 50 in a small RNA sequence database, and still more preferably, the endonuclease is AsiSI, pacI, notI, or bshii.

12. Use of the method according to any one of claims 1-7 or the kit according to any one of claims 8-11 for constructing an RNA sequencing library.

Preferably, the RNA sequencing library is selected from the group consisting of: plasma small RNA sequencing library, CLIP library, RIP library, meRIP library and GRO library.

13. A method of determining sequence information of a small RNA molecule, comprising:

constructing a sequencing library by the method of claim 2 based on a sample of small RNA molecules; sequencing the sequencing library to obtain a sequencing result; and obtaining sequence information of the small RNA molecule based on the sequencing result.

14. A system for determining sequence information of a small RNA molecule, comprising:

the kit according to any one of claims 8-11;

the sequencing device is used for sequencing the sequencing library constructed by the kit on a sample to obtain a sequencing result of the sample; and

and the analysis device is used for analyzing the sequencing result so as to obtain the sequence information of the small RNA molecule.

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