A method for RNA library construction and sequencing

Abstract

The application belongs to the field of nucleic acid sequencing, and specifically discloses a method for constructing an RNA library. The method comprises the following steps: screening mRNA with a 5' end cap, connecting the mRNA with a 5' end adaptor to obtain a connection product, and then using a reverse transcription primer with a single molecule tag sequence to perform reverse transcription on the connection product, and further designing a suitable amplification primer to perform PCR amplification on the adaptor connection product, so as to obtain the RNA library. Compared with the prior art, in the method, the single adaptor is connected to the 5' end of the complete and intact RNA, the specific first-strand cDNA is synthesized through reverse transcription, the synthesis of the second-strand cDNA is not required, other breaking and digestion processes are not required, and the chain specificity is directly achieved, so that the operation is simple, and a large amount of working hours is saved. The method can reduce the requirement for the initial amount of the sample, reduce the cost of library construction, simplify the detection process, and save the operation time.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid sequencing, specifically relating to a method for constructing and sequencing an RNA library. Background Technology

[0002] Today, transcriptome sequencing technology is widely used in various fields such as healthcare and life sciences, and gene expression can be measured through high-throughput sequencing of the transcriptome. Through multiple regulatory mechanisms, various different RNA 5' ends are generated during and after transcription, and the variable transcription start site (TSS) of a single gene can produce transcript isoforms with different functions. The occurrence of many diseases is related to changes in the expression of different transcript isoforms of human genes or pathogen genes. Variable 5' ends may lead to differences in the first exon of a transcript, resulting in different N-terminal protein isoforms, or differences in the length of the 5'-UTR, thus affecting the binding of regulatory factors within the 5'-UTR region or the translation of ORFs. Furthermore, since transcriptional regulatory elements such as promoters are often located near the transcription start site, precise localization of the transcription start site helps to discover variable transcription factor binding sites. This transcription may be regulated by different promoters and enhancers, and under different physiological conditions, it may selectively express transcription factors based on chromatin state and transcription factor expression.

[0003] Currently, different transcriptomic isoforms can be distinguished using third-generation full-length transcriptome sequencing such as PacBio Iso-seq and Nanopore. However, these technologies cannot eliminate random 5' ends caused by RNA degradation or breaks at specific structural locations, resulting in significant background noise in the localization of transcription start sites. Additionally, techniques such as 5'-RACE (5'-rapid amplification of cDNA ends cDNA), CAGE (cap analysis of gene expression), and second-generation mRNA-seq can also be used to distinguish different transcriptomic isoforms to some extent. Specifically, 5'-RACE involves digesting and removing DNA, rRNA, and other RNA, as well as degraded or broken mRNA residues, to purify full-length mRNA. The cap structure of the full-length RNA is then removed, and a fixed adapter sequence is ligated. PCR amplifies the relevant transcripts of a specific gene, and first-generation sequencing obtains the 5' end information of that gene. While this technique can purify mRNA with 5'-cap structures, it only performs PCR amplification of a specific gene and, as verified by Sanger sequencing, cannot obtain complete transcriptomic information. CAGE technology: This technique involves injecting a large amount of RNA and enriching the 5'-cap structure with biotin labeling. Then, it uses random primers for reverse transcription to the 5' end of the RNA, and adapters are ligated to both ends of the cDNA for library construction. Sequencing is then performed using a second-generation short-read sequencing platform. However, this technique requires a large amount of RNA and the experimental steps are cumbersome (taking more than a week). Second-generation mRNA-seq and third-generation PacBio Iso-seq technologies: Both of these technologies use reverse transcription primers containing Oligo dT or random primers for reverse transcription. The resulting cDNA amplification product is then obtained by PCR. Sequencing is performed by adding adapters to fragmented RNA and then using a second-generation platform, or by adding adapters to the full-length cDNA and then amplifying it before sequencing on a third-generation platform. These methods provide relatively accurate gene expression quantification or relatively accurate transcript isoform detection, respectively. However, second-generation mRNA-seq cannot accurately locate the transcription start information at the 5' end of the RNA, and PacBio Iso-seq reverse transcription cannot eliminate interference noise from fragmented RNA.

[0004] Therefore, there is an urgent need for a method that can accurately locate the transcription start site at the 5' end of a single mRNA molecule at low cost, and accurately distinguish and quantify different transcript subtypes of a single gene. Summary of the Invention

[0005] The first aspect of this invention aims to provide a method for constructing an RNA library.

[0006] The second aspect of this invention is to provide a nucleic acid library.

[0007] A third aspect of the present invention is to provide a sequencing method.

[0008] The fourth aspect of this invention aims to provide the application of the method for constructing the RNA library of the first aspect of this invention, the nucleic acid library of the second aspect of this invention, or the sequencing method of the third aspect of this invention.

[0009] The fifth aspect of this invention is to provide a nucleic acid composition.

[0010] The sixth aspect of this invention aims to provide a reagent combination for 5' RNA linker ligation-reverse transcription.

[0011] The seventh aspect of this invention aims to provide a library construction kit.

[0012] The object of the eighth aspect of the present invention is to provide a method.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A first aspect of the present invention provides a method for constructing an RNA library, comprising the following steps:

[0015] Screen for mRNAs with 5' end caps;

[0016] The 5' end of the mRNA is ligated to the 5' RNA adapter to obtain the ligation product;

[0017] The ligation product was reverse transcribed using reverse transcription primers to obtain first-strand cDNA.

[0018] The first-strand cDNA was amplified by PCR using 3' end primers and RNA adapter primers to obtain an RNA library.

[0019] The reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0020] Adding single-molecule tags significantly reduces the impact of the PCR process on quantification, lowers the difficulty of library preparation, and reduces the limitations on the amount of RNA input.

[0021] In some embodiments of the present invention, the amplification binding sequence in the reverse transcription primer is at least partially identical to that of the 3' end primer.

[0022] In some embodiments of the present invention, the 3' primer includes a 5' end modification.

[0023] In some embodiments of the present invention, the 5' end modification includes at least one of phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl modification, or 3'-O-azidomethyl modification.

[0024] In some embodiments of the present invention, the 5' RNA adapter is a single linker.

[0025] In some embodiments of the present invention, the 5' RNA adapter is a universal sequence.

[0026] In some embodiments of the present invention, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide (i.e., rNTP, such as rATP, rGTP, rTP, rUTP).

[0027] In some embodiments of the present invention, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides (rNTPs) and deoxyribonucleotides (dNTPs).

[0028] In some embodiments of the present invention, the RNA adapter primer is at least partially reverse complementary to the 3' end of the 5' RNA adapter.

[0029] In some embodiments of the present invention, the single-molecule tag sequence includes a unique molecular tag (UMI, used to count the copy number of nucleic acid molecules in a sample) sequence and a sample tag sequence (used to distinguish different samples for subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence).

[0030] In some embodiments of the present invention, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base.

[0031] In some embodiments of the present invention, the proportion of each A, T, G or C base in the N base sequence is 20% to 30%.

[0032] In some embodiments of the present invention, the length of the unique molecular tag sequence is 4 to 20 bp.

[0033] In some embodiments of the present invention, the unique molecular tag sequence does not exhibit complementary or reverse complementary binding with any primers used in library construction or sequencing, or with any sequences within the tag sequence itself. For example, if a portion of the unique molecular tag sequence is ATGG, then no primers used in library construction or sequencing, or any sequences within the tag sequence itself, contain TACC and / or CCAT. Furthermore, the unique molecular tag sequence does not form hairpin structures (stem-loop structures), palindromic structures, dimers, etc., with other sequences (such as any primers used in library construction or sequencing, or any sequences within the tag sequence itself).

[0034] In some embodiments of the present invention, the sample tag sequence is located at the 3' end of the unique molecular tag sequence.

[0035] In some embodiments of the present invention, the length of the sample label sequence is 4 to 20 bp.

[0036] By screening 5'-cap structure mRNAs as initial input samples for library construction, the 5' start site of mRNA can be better located, new transcripts can be discovered, and background noise caused by broken mRNAs during sequencing can be removed.

[0037] In some embodiments of the present invention, the reverse transcription primers include, from the 5'-3' end, an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0038] In some embodiments of the present invention, the mRNA is pretreated before the 5' end of the mRNA is linked to the 5' RNA adapter.

[0039] In some embodiments of the present invention, the pretreatment includes purification of mRNA with a 5' cap structure and 5'-decap treatment. The present invention obtains the precise location of the mRNA transcription start site by purifying mRNA with a 5'-cap structure and attaching a linker to its 5' end.

[0040] In some embodiments of the present invention, the purification process of the 5'-cap mRNA includes the step of treating the RNA with at least one of RNA polyphosphatase, exonuclease, CIP enzyme, endonuclease or RNase inhibitor.

[0041] In some embodiments of the present invention, the RNA polyphosphatase includes at least one of aluminum-inducible RNA 5' polyphosphatase, Escherichia coli RNA 5' polyphosphatase I, or Shigella RNA 5' polyphosphatase I.

[0042] In some embodiments of the present invention, the endonuclease includes at least one of deoxyribonuclease DNase I, deoxyribonuclease DNase II, deoxyribonuclease, ribonuclease, micrococcal nuclease, endonuclease dsDNase, salt-active endonuclease SAN, or endonuclease Vvn.

[0043] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0044] In some embodiments of the present invention, the 5'-decap treatment includes treating 5'-decap RNA with RNA 5' pyrophosphate hydrolase (RppH) to remove the 5' cap structure, leaving a single phosphate group.

[0045] In some embodiments of the present invention, the system in which the 5' end of mRNA is linked to a 5' RNA adapter includes RNA, a 5' RNA adapter, a ligase, an RNase inhibitor, and a buffer.

[0046] In some embodiments of the present invention, the ligase includes at least one of E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), or a truncated form of T4 RNA ligase 2.

[0047] In some embodiments of the present invention, the ligase is T4 RNA ligase, which facilitates the ligation of RNA adapters at the 5' end of single-stranded nucleic acid molecules, resulting in high ligation efficiency and low cost.

[0048] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0049] In some embodiments of the present invention, the RNase inhibitor is SUPERase In.

[0050] In some embodiments of the present invention, the concentration of SUPERase In in the system is 0.01 v / v% to 1 v / v%.

[0051] In some embodiments of the present invention, the buffer solution includes at least one of dNTPs, enhancers, stabilizers, or metal ions.

[0052] In some embodiments of the present invention, the buffer solution comprises adenine nucleoside triphosphate (ATP) and / or guanosine triphosphate (GTP).

[0053] In some embodiments of the present invention, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween.

[0054] In some embodiments of the present invention, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO.

[0055] In some embodiments of the present invention, the stabilizer and the reinforcing agent may be selected as the same, partially the same, or different substances.

[0056] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0057] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0058] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0059] In some embodiments of the present invention, the concentration of the 3' RNA adapter in the system is 1–20 μM.

[0060] In some embodiments of the present invention, the concentration of the ligase in the system is 0.1–10 U / μL.

[0061] In some embodiments of the present invention, the concentration of the reinforcing agent in the system is 0.01 to 1 v / v%.

[0062] In some embodiments of the present invention, the concentration of the stabilizer in the system is 0.01 to 0.5 μg / μL.

[0063] In some embodiments of the present invention, the ligation product is purified before reverse transcription.

[0064] In some embodiments of the present invention, the purification method includes magnetic bead purification, centrifugal column purification, or a combination thereof.

[0065] In some embodiments of the present invention, the magnetic bead purification includes the following steps: mixing the ligation product with the capture probe, reacting, mixing with the magnetic beads, and performing magnetic adsorption to obtain the purified ligation product.

[0066] In some embodiments of the present invention, the nucleotide sequence of the capture probe is at least partially identical to the RNA adapter sequence.

[0067] In some embodiments of the present invention, the concentration of the capture probe in the reaction system is 5–20 μM.

[0068] In some embodiments of the present invention, the reaction conditions are: incubation at 70–90°C for 5–20 minutes, followed by slow cooling to room temperature.

[0069] In some embodiments of the present invention, the reverse transcription system includes reverse transcriptase, reverse transcription primers, and buffer solution.

[0070] In some embodiments of the present invention, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof.

[0071] In some embodiments of the present invention, the reverse transcriptase includes at least one of SmartScribe reverse transcriptase, MaximaHMinus reverse transcriptase, SuperscriptII reverse transcriptase, SuperscriptIII reverse transcriptase, or Alpha reverse transcriptase.

[0072] In some embodiments of the present invention, the concentration of the reverse transcriptase in the system is 0.1–10 U / μL.

[0073] In some embodiments of the present invention, the concentration of the reverse transcription primer in the system is 0.1–20 μM.

[0074] In some embodiments of the present invention, the buffer solution includes dNTPs, metal ions, or combinations thereof.

[0075] In some embodiments of the present invention, the buffer solution comprises adenine nucleoside triphosphate (ATP) and / or guanosine triphosphate (GTP).

[0076] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0077] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0078] In some embodiments of the present invention, the concentration of dNTPs in the buffer solution in the system is 0.05–5 mM.

[0079] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0080] In some embodiments of the present invention, the reverse transcription process involves selecting a corresponding reverse transcription reaction procedure depending on the transcription reagent used. For details, please refer to commercially available conventional reverse transcription kits and their instructions.

[0081] In some embodiments of the present invention, the reaction system for PCR amplification of the first strand cDNA includes a 3' end primer, an RNA adapter sequence primer, a polymerase, and a PCR enhancer.

[0082] In some embodiments of the present invention, the concentration of the 3' end primer in the reaction system is 0.1 to 1 μM.

[0083] In some embodiments of the present invention, the concentration of the RNA adapter sequence primer in the reaction system is 0.1–1 μM.

[0084] In some embodiments of the present invention, the polymerase includes at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment), T7 DNA polymerase, DNA polymerase I, KAPA HiFi DNA polymerase, or Phusion DNA polymerase.

[0085] In some embodiments of the present invention, the DNA polymerase is selected from Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase, or Pfu DNA polymerase. This is to achieve efficient amplification.

[0086] In some embodiments of the present invention, the concentration of the polymerase in the reaction system is 0.5–2 U / μL.

[0087] In some embodiments of the present invention, the PCR enhancer includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween; preferably betaine, which improves the smooth passage of DNA polymerase through certain complex secondary structures of DNA, prevents DNA polymerase from dissociating from the template DNA, helps DNA polymerase extend along the DNA template, and eliminates the dependence of denaturation temperature on bases.

[0088] In some embodiments of the present invention, the concentration of the PCR enhancer in the reaction system is 0.5–2 M.

[0089] A second aspect of the present invention provides a nucleic acid library constructed by the method of the first aspect of the present invention.

[0090] A third aspect of the present invention provides a sequencing method comprising the step of sequencing a nucleic acid library according to a second aspect of the present invention.

[0091] After obtaining a cDNA library containing complete 5' end information of the RNA, further testing can be performed using any conventional sequencing method.

[0092] In some embodiments of the present invention, the sequencing method includes at least one of PacBio, Nanopore, Cyclone, or DNB sequencing.

[0093] Long-read sequencing, such as SE600 / 800, can directly sequence the 5' end ORF and UTR regions of transcripts, and their single-molecule tags can be used to achieve accurate differentiation and quantification of different transcripts.

[0094] In some embodiments of the present invention, the sequencing method is DNB sequencing.

[0095] Preferably, the DNB sequencing includes any one of a1) to a2):

[0096] a1) Nucleic acid was subjected to cyclization, reverse controlled extension, end ligation, cyclization, digestion of linear single strands, purification, and rolling circle replication in sequence to obtain DNA nanospheres;

[0097] a2) The reverse controlled extension, end ligation, cyclization reaction and rolling circle replication of nucleic acid are completed in one step to obtain DNA nanospheres.

[0098] In some embodiments of the present invention, the sequencing process further includes the following step: library quality control.

[0099] In some embodiments of the present invention, the length of the product obtained by the controlled extension is 300 to 1500 bp.

[0100] By using reverse control extension technology, the detection efficiency of the 5' end sequence can be improved while accurately quantifying each transcript subtype of a gene, which helps to detect 5' end transcriptional regulatory elements more sensitively.

[0101] A fourth aspect of the invention provides the application of b1) to b3) in c1) to c7):

[0102] b1) The RNA library in the first aspect of the present invention;

[0103] b2) The nucleic acid library of the second aspect of the present invention;

[0104] b3) The sequencing method of the third aspect of the present invention;

[0105] c1) Detect / locate the 5' transcription start site of RNA;

[0106] c2) Detect the 5' UTR of RNA;

[0107] c3) Detect the 5' end ORF of RNA;

[0108] c4) Distinguish between different transcript subtypes of a single gene;

[0109] c5) Quantitative detection of different transcript subtypes;

[0110] c6) gene expression regulation and function study;

[0111] c7) Gene editing (such as CRISPR / Cas systems).

[0112] A fifth aspect of the present invention provides a nucleic acid composition comprising a 5' RNA adapter and a reverse transcription primer.

[0113] In some embodiments of the present invention, the 5' RNA adapter is a single linker.

[0114] In some embodiments of the present invention, the 5' RNA adapter is a universal sequence.

[0115] In some embodiments of the present invention, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide (i.e., rNTP, such as rATP, rGTP, rCTP, rUTP).

[0116] In some embodiments of the present invention, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides (rNTPs) or deoxyribonucleotides (dNTPs).

[0117] In some embodiments of the present invention, the reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0118] In some embodiments of the present invention, the single-molecule tag sequence includes a unique molecular tag (UMI, used to count the copy number of nucleic acid molecules in a sample) sequence and a sample tag sequence (used to distinguish different samples for subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence).

[0119] In some embodiments of the present invention, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base.

[0120] In some embodiments of the present invention, the proportion of each A, T, G or C base in the N base sequence is 20% to 30%.

[0121] In some embodiments of the present invention, the length of the unique molecular tag sequence is 4 to 20 bp.

[0122] In some embodiments of the present invention, the unique molecular tag sequence does not exhibit complementary or reverse complementary binding with any primers used in library construction or sequencing, or with any sequences within the tag sequence itself. For example, if a portion of the unique molecular tag sequence is ATGG, then no primers used in library construction or sequencing, or any sequences within the tag sequence itself, contain TACC and / or CCAT. Furthermore, the unique molecular tag sequence does not form hairpin structures (stem-loop structures), palindromic structures, dimers, etc., with other sequences (such as any primers used in library construction or sequencing, or any sequences within the tag sequence itself).

[0123] In some embodiments of the present invention, the sample tag sequence is located at the 3' end of the unique molecular tag sequence.

[0124] In some embodiments of the present invention, the length of the sample label sequence is 4 to 20 bp.

[0125] In some embodiments of the present invention, the reverse transcription primers include, from the 5'-3' end, an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0126] A sixth aspect of the present invention provides a reagent combination for 5' RNA linker ligation-reverse transcription, comprising the nucleic acid composition of the fifth aspect of the present invention, a ligase, and a reverse transcriptase.

[0127] In some embodiments of the present invention, the ligase can catalyze the formation of phosphodiester bonds between the 5'-P end and the 3'-OH end of a single-stranded oligonucleotide or mononucleotide, either intermolecularly or intramolecularly.

[0128] In some embodiments of the present invention, the ligase includes at least one selected from E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), or a truncated form of T4 RNA ligase 2.

[0129] In some embodiments of the present invention, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof.

[0130] In some embodiments of the present invention, the reverse transcriptase includes at least one of SmartScribe reverse transcriptase, MaximaHMinus reverse transcriptase, SuperscriptII reverse transcriptase, SuperscriptIII reverse transcriptase, or Alpha reverse transcriptase.

[0131] In some embodiments of the present invention, the reagent combination further includes a buffer solution.

[0132] In some embodiments of the present invention, the buffer solution includes at least one of NTP, enhancer, stabilizer, metal ion or inhibitor.

[0133] In some embodiments of the present invention, the buffer solution comprises adenine nucleoside triphosphate (ATP) and / or guanosine triphosphate (GTP).

[0134] In some embodiments of the present invention, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween.

[0135] In some embodiments of the present invention, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO.

[0136] In some embodiments of the present invention, the stabilizer and the reinforcing agent may be selected as the same, partially the same, or different substances.

[0137] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0138] In some embodiments of the present invention, the inhibitor includes an RNase inhibitor.

[0139] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0140] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0141] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0142] A seventh aspect of the present invention provides a library construction kit comprising the nucleic acid composition of the fifth aspect of the present invention and / or the reagent combination of the sixth aspect of the present invention.

[0143] In some embodiments of the present invention, the library construction kit further includes a DNB preparation reagent.

[0144] In some embodiments of the present invention, the DNB preparation reagent comprises a polynucleotide kinase.

[0145] In some embodiments of the present invention, the polynucleotide kinase is T4 polynucleotide kinase.

[0146] In some embodiments of the present invention, the DNB preparation reagent includes any of the substances involved in any of the following DNB preparation methods:

[0147] a1) Nucleic acid was subjected to cyclization, reverse controlled extension, end ligation, cyclization, digestion of linear single strands, purification, and rolling circle replication in sequence to obtain DNA nanospheres;

[0148] a2) The reverse controlled extension, end ligation, cyclization reaction and rolling circle replication of nucleic acid are completed in one step to obtain DNA nanospheres.

[0149] In some embodiments of the present invention, the DNB preparation reagent includes singletube long fragment read primers and splint oligos.

[0150] In some embodiments of the present invention, the circularization primer includes complementary sequences to the 3' end sequence and the 5' end sequence of the linear sequence. It is used to connect the two ends of the linear sequence and to circularize the linear sequence. Based on this, it can also be used to screen DNA fragments connected to two different adapters, and as a primer for rolling circle amplification (RCA) to prepare DNB.

[0151] In some embodiments of the present invention, the library construction kit further includes exonucleases.

[0152] In some embodiments of the present invention, the exonuclease includes at least one of Exonuclease I, Exonuclease II, or Exonuclease III.

[0153] In some embodiments of the present invention, the library construction kit further includes a polymerase.

[0154] In some embodiments of the present invention, the polymerase includes at least one of T4 DNA polymerase, DNA polymerase I Klenow large fragment, T7 DNA polymerase, DNA polymerase I, KAPAHiFi DNA polymerase, or Phusion DNA polymerase.

[0155] In some embodiments of the present invention, the DNA polymerase is selected from Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase, or Pfu DNA polymerase. This is to achieve efficient amplification.

[0156] In some embodiments of the present invention, the library construction kit includes a buffer solution.

[0157] In some embodiments of the present invention, the buffer solution includes at least one of dNTPs, metal ions, or inhibitors.

[0158] In some embodiments of the present invention, the buffer solution comprises adenine nucleoside triphosphate (ATP) and / or guanosine triphosphate (GTP).

[0159] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0160] In some embodiments of the present invention, the inhibitor includes an RNase inhibitor.

[0161] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0162] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0163] An eighth aspect of the invention provides a method comprising the step of measuring RNA to be tested using the sequencing method of the third aspect of the invention; or

[0164] The step includes processing the RNA to be tested using the nucleic acid composition of the fifth aspect of the present invention, the reagent combination of the sixth aspect of the present invention, or the library construction kit of the seventh aspect of the present invention;

[0165] The method includes any one of d1) to d5):

[0166] d1) Methods for detecting / locating the 5' transcription start site of RNA;

[0167] d2) Methods for detecting the 5' UTR of RNA;

[0168] d3) Methods for detecting the 5' end ORF of RNA;

[0169] d4) Methods for distinguishing different transcriptomic subtypes of a single gene;

[0170] d5) Methods for quantitative detection of different transcript subtypes.

[0171] The beneficial effects of this invention are:

[0172] This invention obtains a ligation product by ligating RNA to its 5' adapter, then reverse transcription of the ligation product using reverse transcription primers with a single-molecule tag sequence, and further amplification using appropriately designed amplification primers to perform PCR amplification of the adapter ligation product, thus obtaining an RNA library. Compared with existing methods, this method simplifies the operation and saves a significant amount of time because the single-linker ligates a completely intact 5' RNA, synthesizes specific first-strand cDNA through reverse transcription, and eliminates the need for second-strand cDNA synthesis and other digestion processes, directly achieving strand specificity. This method reduces the required starting sample volume, lowers the cost of library construction, simplifies the detection process, saves operation time, and provides more low-cost and flexible options for clinical applications. Furthermore, it enables precise localization of the 5' transcription start site of a single mRNA molecule on a next-generation sequencing platform at low cost, accurately distinguishes and quantifies different transcript subtypes of a single gene, and better detects the RNA 5' transcriptional regulatory region.

[0173] Furthermore, this invention combines the advantages of single-molecule labeling, reverse controlled extension technology, and 5'-capped RNA purification and adapter ligation technology in 5'-RACE, overcoming the shortcomings of both. For example... Figure 1 As shown: First, non-full-length mRNA and DNA residues are digested using RNA 5'-Polyphosphatase, CIP, Terminator exonuclease, and DNase I. Then, full-length mRNA with a specific marker sequence at the 5' end is obtained through 5'-decapping and linker insertion. cDNA is synthesized using reverse transcription primers with a single-molecule marker, amplified, and then circularized and reverse extended to obtain a library containing precise 5' end sequence information of the full-length mRNA, which is then sequenced. This allows for precise location of transcription start sites, better differentiation and accurate quantification of different transcript subtypes for each gene. Simultaneously, it can detect partial ORF and UTR regions at the 5' end of transcripts. Compared to CAGE technology, this method is simpler to operate and requires less RNA. Circularization and reverse amplification techniques accurately detect the complete 5' end of RNA, avoiding sequencing data waste, reducing costs, and improving the sensitivity and accuracy of detecting binding sites of transcriptional regulatory elements. Attached Figure Description

[0174] Figure 1 This is a schematic diagram of the sequencing library construction method of the present invention.

[0175] Figure 2 This refers to the sequence information during the sequencing library construction process in Embodiment 1 of the present invention.

[0176] Figure 3 This is an agarose gel electrophoresis image of PCR products during the sequencing library construction process in Example 1 of the present invention.

[0177] Figure 4 This document presents a partial data analysis process and results from Embodiment 1 of the present invention; where a represents sequencing quality control data; b represents an example of mapping results; and c represents a statistical example of transcript subtypes showing TSS differences in single genes.

[0178] Figure 5 The detection method described in Example 1 was used to analyze the 5' end of the RNA of hepatitis B virus (HBV) by utilizing the terminal transferase activity of reverse transcriptase. Detailed Implementation

[0179] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0180] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0181] A first aspect of the present invention provides a method for constructing an RNA library, comprising the following steps:

[0182] Screen for mRNAs with 5' caps;

[0183] The 5' end of the mRNA is ligated to the 5' RNA adapter to obtain the ligation product;

[0184] The ligation product was reverse transcribed using reverse transcription primers to obtain first-strand cDNA.

[0185] The first-strand cDNA was amplified by PCR using 3' end primers and RNA adapter primers to obtain an RNA library.

[0186] The reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0187] Adding single-molecule tags significantly reduces the impact of the PCR process on quantification, lowers the difficulty of library preparation, and reduces the limitations on the amount of RNA input.

[0188] The above-described library construction method involves ligating RNA to its 5' adapter, reverse transcription of the ligation product using reverse transcription primers with a single-molecule tag sequence, and PCR amplification of the adapter ligation product using appropriately designed amplification primers to obtain an RNA library. Compared with existing methods, this method simplifies the operation and saves significant time because the single-link adapter ligates specific first-strand cDNA, eliminating the need for second-strand cDNA synthesis and other digestion processes to achieve strand specificity. This method reduces the required sample size, lowers library construction costs, simplifies the detection process, saves operation time, and provides more cost-effective and flexible options for clinical applications. Furthermore, it enables precise localization of the 5' transcription start site of a single mRNA molecule on a next-generation sequencing platform at low cost, accurately distinguishing and quantifying different transcript subtypes of a single gene, and better detecting the RNA 5' transcriptional regulatory region.

[0189] In some embodiments of the present invention, the amplification binding sequence in the reverse transcription primer is at least partially identical to that of the 3' end primer.

[0190] In some embodiments of the present invention, the 3' primer includes a 5' end modification.

[0191] In some embodiments of the present invention, the 5' end modification includes at least one of phosphorylation, phosphorylation analog, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl modification, or 3'-O-azidomethyl modification.

[0192] In some embodiments of the present invention, the 5' RNA adapter is a single linker.

[0193] In some embodiments of the present invention, the 5' RNA adapter is a universal sequence. The universal sequence refers to a known sequence that can match elements such as the multiple cloning site, promoter, and terminator of existing vectors.

[0194] In some embodiments of the present invention, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide (i.e., rNTP, such as rATP, rGTP, tCTP, rUTP).

[0195] In some embodiments of the present invention, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides (rNTPs) or deoxyribonucleotides (dNTPs).

[0196] In some embodiments of the present invention, the RNA adapter primer is at least partially reverse complementary to the 3' end of the 5' RNA adapter.

[0197] In some embodiments of the present invention, the single-molecule tag sequence includes a unique molecular tag (UMI, used to count the copy number of nucleic acid molecules in a sample) sequence and a sample tag sequence (used to distinguish different samples for subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence).

[0198] In some embodiments of the present invention, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base.

[0199] In some embodiments of the present invention, the proportion of each A, T, G or C base in the N base sequence is 20% to 30%.

[0200] In some embodiments of the present invention, the length of the unique molecular tag sequence is 4 to 20 bp.

[0201] In some embodiments of the present invention, the unique molecular tag sequence does not exhibit complementary or reverse complementary binding with any primers used in library construction or sequencing, or with any sequences within the tag sequence itself. For example, if a portion of the unique molecular tag sequence is ATGG, then no primers used in library construction or sequencing, or any sequences within the tag sequence itself, contain TACC and / or CCAT. Furthermore, the unique molecular tag sequence does not form hairpin structures (stem-loop structures), palindromic structures, dimers, etc., with other sequences (such as any primers used in library construction or sequencing, or any sequences within the tag sequence itself).

[0202] In some embodiments of the present invention, the sample tag sequence is located at the 3' end of the unique molecular tag sequence.

[0203] In some embodiments of the present invention, the length of the sample label sequence is 4 to 20 bp.

[0204] In some embodiments of the present invention, the reverse transcription primers include, from the 5'-3' end, an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0205] By screening 5'-cap structure mRNAs as initial input samples for library construction, the 5' start site of mRNA can be better located, new transcripts can be discovered, and background noise caused by broken mRNAs during sequencing can be removed.

[0206] In some embodiments of the present invention, the mRNA is pretreated before the 5' end of the mRNA is linked to the 5' RNA adapter.

[0207] In some embodiments of the present invention, the pretreatment includes purification of mRNA with a 5'-cap structure and 5'-decap treatment. The present invention obtains the precise location of the mRNA transcription start site by purifying mRNA with a 5'-cap structure and attaching a linker to its 5' end.

[0208] In some embodiments of the present invention, the purification process of the 5'-cap mRNA includes the step of treating the RNA with at least one of RNA polyphosphatase, exonuclease, CIP enzyme, endonuclease, and RNase inhibitor.

[0209] In some embodiments of the present invention, the RNA polyphosphatase includes at least one of aluminum-inducible RNA 5' polyphosphatase, Escherichia coli RNA 5' polyphosphatase I, or Shigella RNA 5' polyphosphatase I.

[0210] In some embodiments of the present invention, the endonuclease includes at least one of deoxyribonuclease DNase I, deoxyribonuclease DNase II, deoxyribonuclease, ribonuclease, micrococcal nuclease, endonuclease dsDNase, salt-active endonuclease SAN, or endonuclease Vvn.

[0211] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0212] In some embodiments of the present invention, the 5'-decap treatment includes treating 5'-decap RNA with RNA 5' pyrophosphate hydrolase (RppH) to remove the 5' cap structure, leaving a single phosphate group.

[0213] In some embodiments of the present invention, the system in which the 5' end of mRNA is linked to a 5' RNA adapter includes RNA, a 5' RNA adapter, a ligase, an RNase inhibitor, and a buffer.

[0214] In some embodiments of the present invention, the ligase includes at least one selected from E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circular ligase (ssDNA / RNA CircLigase), or a truncated form of T4 RNA ligase 2.

[0215] In some embodiments of the present invention, the ligase is T4 RNA ligase, which facilitates the ligation of RNA adapters at the 5' end of single-stranded nucleic acid molecules, resulting in high ligation efficiency and low cost.

[0216] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0217] In some embodiments of the present invention, the RNase inhibitor is SUPERase In.

[0218] In some embodiments of the present invention, the concentration of SUPERase In in the system is 0.01 v / v% to 1 v / v%.

[0219] In some embodiments of the present invention, the buffer solution includes at least one of dNTPs, enhancers, stabilizers, or metal ions.

[0220] In some embodiments of the present invention, the buffer solution comprises adenine nucleoside triphosphate (ATP) and / or guanosine triphosphate (GTP).

[0221] In some embodiments of the present invention, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween.

[0222] In some embodiments of the present invention, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO.

[0223] In some embodiments of the present invention, the stabilizer and the reinforcing agent may be selected as the same, partially the same, or different substances.

[0224] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0225] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0226] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0227] In some embodiments of the present invention, the concentration of the 3' RNA adapter in the system is 1–20 μM.

[0228] In some embodiments of the present invention, the concentration of the ligase in the system is 0.1 U / μL to 10 U / μL.

[0229] In some embodiments of the present invention, the concentration of the reinforcing agent in the system is 0.01 v / v% to 1 v / v%.

[0230] In some embodiments of the present invention, the concentration of the stabilizer in the system is 0.01 to 0.5 μg / μL.

[0231] In some embodiments of the present invention, the concentration of the dNTP in the system is 0.05–5 mM.

[0232] In some embodiments of the present invention, the concentration of ATP and / or GTP in the system is 0.1 to 10 mM.

[0233] In some embodiments of the present invention, the ligation product is purified prior to reverse transcription.

[0234] In some embodiments of the present invention, the purification method includes magnetic bead purification, centrifugal column purification, or a combination thereof.

[0235] In some embodiments of the present invention, the magnetic bead purification includes the following steps: mixing the ligation product with the capture probe, reacting, mixing with the magnetic beads, and performing magnetic adsorption to obtain the purified ligation product.

[0236] In some embodiments of the present invention, the nucleotide sequence of the capture probe is at least partially identical to the RNA adapter sequence.

[0237] In some embodiments of the present invention, the concentration of the capture probe in the reaction system is 5–20 μM.

[0238] In some embodiments of the present invention, the reaction conditions are: incubation at 70–90°C for 5–20 minutes, followed by slow cooling to room temperature.

[0239] In some embodiments of the present invention, the reverse transcription system includes reverse transcriptase, reverse transcription primers, and buffer solution.

[0240] In some embodiments of the present invention, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof.

[0241] In some embodiments of the present invention, the reverse transcriptase includes at least one of SmartScribe reverse transcriptase, MaximaHMinus reverse transcriptase, SuperscriptII reverse transcriptase, SuperscriptIII reverse transcriptase, or Alpha reverse transcriptase.

[0242] In some embodiments of the present invention, the concentration of the reverse transcriptase in the system is 0.1–10 U / μL.

[0243] In some embodiments of the present invention, the concentration of the reverse transcription primer in the system is 0.1–20 μM.

[0244] In some embodiments of the present invention, the buffer solution comprises dNTPs and metal ions, and at least one of ATP or GTP.

[0245] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0246] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0247] In some embodiments of the present invention, the concentration of the dNTP in the system is 0.05–5 mM.

[0248] In some embodiments of the present invention, the concentration of ATP and / or GTP in the system is 0.1 to 10 mM.

[0249] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion; more specifically, magnesium ion.

[0250] In some embodiments of the present invention, the concentration of metal ions in the buffer solution in the system is 0.1 to 50 mM.

[0251] In some embodiments of the present invention, the reverse transcription process involves selecting a corresponding reverse transcription reaction procedure depending on the transcription reagent used. For details, please refer to commercially available conventional reverse transcription kits and their instructions.

[0252] In some embodiments of the present invention, the reaction system for PCR amplification of the first strand cDNA includes a 3' end primer, an RNA adapter sequence primer, a polymerase, and a PCR enhancer.

[0253] In some embodiments of the present invention, the concentration of the 3' end primer in the reaction system is 0.1 to 1 μM.

[0254] In some embodiments of the present invention, the concentration of the RNA adapter sequence primer in the reaction system is 0.1–1 μM.

[0255] In some embodiments of the present invention, the polymerase includes at least one of T4 DNA polymerase, DNA polymerase I (Klenow) large fragment (Klenow fragment), T7 DNA polymerase, DNA polymerase I, KAPA HiFi DNA polymerase, or Phusion DNA polymerase.

[0256] In some embodiments of the present invention, the DNA polymerase is selected from Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase, or Pfu DNA polymerase. This is to achieve efficient amplification.

[0257] In some embodiments of the present invention, the concentration of the polymerase in the reaction system is 0.5–2 U / μL.

[0258] In some embodiments of the present invention, the PCR enhancer includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween; preferably betaine, which improves the smooth passage of DNA polymerase through certain complex secondary structures of DNA, prevents DNA polymerase from dissociating from the template DNA, helps DNA polymerase extend along the DNA template, and eliminates the dependence of denaturation temperature on bases.

[0259] In some embodiments of the present invention, the concentration of the PCR enhancer in the reaction system is 0.5–2 M.

[0260] A second aspect of the present invention provides a nucleic acid library constructed by the method of the first aspect of the present invention.

[0261] Based on this nucleic acid library, it is possible to accurately locate the 5' transcription start site of a single mRNA molecule on a next-generation sequencing platform at low cost, accurately distinguish and quantify different transcript subtypes of a single gene, and better detect the RNA 5' transcriptional regulatory region.

[0262] A third aspect of the present invention provides a sequencing method comprising the step of sequencing a nucleic acid library according to a second aspect of the present invention.

[0263] After obtaining a cDNA library containing complete 5' end information of the RNA, further testing can be performed using any conventional sequencing method.

[0264] In some embodiments of the present invention, the sequencing method includes at least one of PacBio, Nanopore, Cyclone, or DNB sequencing.

[0265] Long-read sequencing, such as SE600 / 800, can directly sequence the 5' end ORF and UTR regions of transcripts, and their single-molecule tags can be used to achieve accurate differentiation and quantification of different transcripts.

[0266] In some embodiments of the present invention, the sequencing method is DNB sequencing.

[0267] Preferably, the DNB sequencing includes any one of a1) to a2):

[0268] a1) The DNA nanospheres were obtained by sequentially performing cyclization, controlled extension, end joining, cyclization, digestion of linear single strands, purification, and rolling circle replication.

[0269] a2) Controlled extension, end-joining, cyclization reaction and rolling circle replication are completed in one step to obtain DNA nanospheres.

[0270] In some embodiments of the present invention, the sequencing process further includes the following step: library quality control.

[0271] In some embodiments of the present invention, the length of the product obtained by the controlled extension is 300 to 1500 bp.

[0272] The sequencing method provided by this invention can accurately locate the transcription start site at the 5' end of a single mRNA molecule at low cost, accurately distinguish and quantify different transcript isotypes of a single gene, and better detect the RNA 5' transcription regulatory region. The main technical solution process is as follows:

[0273] 1) Collect total RNA and digest it with RNA 5'-Polyphosphatase, CIP, Terminator exonuclease, and DNase I to remove incomplete mRNA and other RNA, or any monophosphate groups that may be present at the 5' end, as well as DNA residues;

[0274] 2) Remove the 5' cap structure by RppH enzyme treatment, leaving a single phosphate group;

[0275] 3) Ligate the RNA adapter to the 5' end of the RNA;

[0276] 4)(Optional) Purify full-length mRNA with adapters using a biotin-capture sequence and Strepavidin C1 magnetic beads;

[0277] 5) Reverse transcription is performed using reverse transcription primers with single-molecule tags;

[0278] 6) Perform PCR amplification of cDNA for 10-12 cycles using PCR primers with RNA 5' adapter sequences;

[0279] 7) The product from step 6) is denatured, circularized, and digested to obtain single-stranded circular DNA molecules;

[0280] 8) Control the input of dNTPs and extend the product from 7) to a certain length starting from the connector end;

[0281] 9) Connect the product from 8) to the second adapter and use it as a template for PCR amplification for 8 more cycles;

[0282] 10) The product from step 9) is denatured, circularized, and digested again to obtain single-stranded circular DNA molecules;

[0283] 11) Prepare DNB and sequence it using the MGI next-generation sequencing platform (PE150 / SE600).

[0284] By selecting RNA with a 5'-cap structure as the initial input sample for library construction, the 5' start site of RNA can be better located, new transcripts can be discovered, and background noise caused by broken RNA during sequencing can be removed. Adding single-molecule tags significantly reduces the impact of PCR on quantification, simplifies library construction, and reduces limitations on the amount of RNA input. Reverse controlled extension technology improves the detection efficiency of the 5' end sequence while accurately quantifying each transcript subtype, facilitating more sensitive detection of 5' end transcriptional regulatory elements. Circularization and reverse amplification techniques accurately detect the intact 5' end of RNA, avoiding sequencing data waste, reducing costs, and further improving the sensitivity and accuracy of detecting binding sites for transcriptional regulatory elements.

[0285] Further control of the extended fragment length, combined with MGI's current longer read sequencing methods such as SE600, can yield longer read libraries and sequencing results with better coverage and accuracy; or the extension control step can be removed, and the full length of the PCR 1 product can be sequenced directly using PacBio, Nanopore, or Cyclone full-length sequencing technologies.

[0286] A fourth aspect of the invention provides the application of b1) to b3) in c1) to c7):

[0287] b1) The RNA library of the first aspect of the present invention; b2) The nucleic acid library of the second aspect of the present invention; b3) The sequencing method of the third aspect of the present invention; c1) Detection / localization of the transcription start site at the 5' end of RNA; c2) Detection of the UTR at the 5' end of RNA; c3) Detection of the ORF at the 5' end of RNA; c4) Differentiation of different transcript subtypes of a single gene; c5) Quantitative detection of different transcript subtypes; c6) Gene expression regulation and functional studies; c7) Gene editing (such as CRISPR / Cas system).

[0288] A fifth aspect of the present invention provides a nucleic acid composition comprising a 5' RNA adapter and a reverse transcription primer.

[0289] In some embodiments of the present invention, the 5' RNA adapter is a single linker.

[0290] In some embodiments of the present invention, the 5' RNA adapter is a universal sequence.

[0291] In some embodiments of the present invention, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide (i.e., rNTP, such as rATP, rGTP, rCTP, rUTP).

[0292] In some embodiments of the present invention, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides (rNTPs) and deoxyribonucleotides (dNTPs).

[0293] In some embodiments of the present invention, the reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

[0294] In some embodiments of the present invention, the single-molecule tag sequence includes a unique molecular tag (UMI, used to count the copy number of nucleic acid molecules in a sample) sequence and a sample tag sequence (used to distinguish different samples for subsequent multi-sample mixed sequencing. For example, it can be a barcode sequence or an index sequence).

[0295] In some embodiments of the present invention, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base.

[0296] In some embodiments of the present invention, the proportion of each A, T, G or C base in the N base sequence is 20% to 30%.

[0297] In some embodiments of the present invention, the length of the unique molecular tag sequence is 4 to 20 bp.

[0298] In some embodiments of the present invention, the unique molecular tag sequence does not exhibit complementary or reverse complementary binding with any primers used in library construction or sequencing, or with any sequences within the tag sequence itself. For example, if a portion of the unique molecular tag sequence is ATGG, then no primers used in library construction or sequencing, or any sequences within the tag sequence itself, contain TACC and / or CCAT. Furthermore, the unique molecular tag sequence does not form hairpin structures (stem-loop structures), palindromic structures, dimers, etc., with other sequences (such as any primers used in library construction or sequencing, or any sequences within the tag sequence itself).

[0299] In some embodiments of the present invention, the sample tag sequence is located at the 3' end of the unique molecular tag sequence.

[0300] In some embodiments of the present invention, the length of the sample label sequence is 4 to 20 bp.

[0301] An RNA library can be obtained by reverse transcription of the ligation product using reverse transcription primers with a single-molecule tag sequence, followed by PCR amplification of the ligation product using suitable amplification primers. Using the aforementioned nucleic acid reagents reduces the required initial sample volume, lowers the cost of library construction, simplifies the detection process, and saves operation time.

[0302] A sixth aspect of the present invention provides a reagent combination for 5' RNA linker ligation-reverse transcription, comprising the nucleic acid composition of the fifth aspect of the present invention, a ligase, and a reverse transcriptase.

[0303] In some embodiments of the present invention, the ligase can catalyze the formation of phosphodiester bonds between the 5'-P end and the 3'-OH end of a single-stranded oligonucleotide or mononucleotide, either intermolecularly or intramolecularly.

[0304] In some embodiments of the present invention, the ligase comprises at least one of E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circularization ligase (ssDNA / RNACircLigase), or a truncated form of T4 RNA ligase 2.

[0305] In some embodiments of the present invention, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof.

[0306] In some embodiments of the present invention, the reverse transcriptase includes at least one of SmartScribe reverse transcriptase, MaximaHMinus reverse transcriptase, SuperscriptII reverse transcriptase, SuperscriptIII reverse transcriptase, or Alpha reverse transcriptase.

[0307] In some embodiments of the present invention, the reagent combination further includes a buffer solution.

[0308] In some embodiments of the present invention, the buffer solution includes at least one of dNTPs, enhancers, stabilizers, metal ions, or inhibitors.

[0309] In some embodiments of the present invention, the buffer solution comprises ATP and / or GTP.

[0310] In some embodiments of the present invention, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween.

[0311] In some embodiments of the present invention, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO.

[0312] In some embodiments of the present invention, the stabilizer and the reinforcing agent may be selected as the same, partially the same, or different substances.

[0313] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0314] In some embodiments of the present invention, the inhibitor includes an RNase inhibitor.

[0315] In some embodiments of the present invention, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin.

[0316] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0317] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0318] A seventh aspect of the present invention provides a library construction kit comprising the nucleic acid composition of the fifth aspect of the present invention and / or the reagent combination of the sixth aspect of the present invention.

[0319] In some embodiments of the present invention, the library construction kit further includes a DNB preparation reagent.

[0320] In some embodiments of the present invention, the DNB preparation reagent comprises a polynucleotide kinase.

[0321] In some embodiments of the present invention, the polynucleotide kinase is T4 polynucleotide kinase.

[0322] In some embodiments of the present invention, the DNB preparation reagent comprises any of the substances involved in any of the following DNB preparation methods:

[0323] a1) The DNA nanospheres were obtained by sequentially performing cyclization, controlled extension, end joining, cyclization, digestion of linear single strands, purification, and rolling circle replication.

[0324] a2) Controlled extension, end-joining, cyclization reaction and rolling circle replication are completed in one step to obtain DNA nanospheres.

[0325] In some embodiments of the present invention, the DNB preparation reagent includes singletube long fragment read primers and splint oligos.

[0326] In some embodiments of the present invention, the circularization primer includes complementary sequences to the 3' end sequence and the 5' end sequence of the linear sequence. It is used to connect the two ends of the linear sequence and to circularize the linear sequence. Based on this, it can also be used to screen DNA fragments connected to two different adapters, and as a primer for rolling circle amplification (RCA) to prepare DNB.

[0327] In some embodiments of the present invention, the library construction kit further includes exonucleases.

[0328] In some embodiments of the present invention, the exonuclease includes at least one of Exonuclease I, Exonuclease II, or Exonuclease III.

[0329] In some embodiments of the present invention, the library construction kit further includes a polymerase.

[0330] In some embodiments of the present invention, the polymerase includes at least one of T4 DNA polymerase, DNA polymerase I Klenow large fragment, T7 DNA polymerase, DNA polymerase I, KAPAHiFi DNA polymerase, or Phusion DNA polymerase.

[0331] In some embodiments of the present invention, the DNA polymerase is selected from Q5 hot-start ultra-fidelity DNA polymerase, KAPA hot-start ultra-fidelity DNA polymerase, Platinum hot-start ultra-fidelity DNA polymerase, or Pfu DNA polymerase. This is to achieve efficient amplification.

[0332] In some embodiments of the present invention, the library construction kit includes a buffer solution.

[0333] In some embodiments of the present invention, the buffer solution includes at least one of dNTPs, metal ions, or inhibitors.

[0334] In some embodiments of the present invention, the buffer solution comprises ATP and / or GTP.

[0335] In some embodiments of the present invention, the buffer solution includes at least one of citrate buffer, MES buffer solution, phosphate buffer, Bis-tris buffer or Tris hydrochloric acid buffer.

[0336] In some embodiments of the present invention, the metal ion includes at least one of magnesium ion, manganese ion or calcium ion.

[0337] In some embodiments of the present invention, the inhibitor includes an RNase inhibitor.

[0338] In some embodiments of the present invention, the pH of the buffer solution is 6 to 8.

[0339] Using the above-mentioned kit to construct nucleic acid libraries reduces the initial sample volume requirements and the cost of library construction. It also simplifies the detection process, saves operation time, and provides more low-cost and flexible options for clinical applications. Furthermore, based on this, it enables precise localization of the 5' transcription start site of a single mRNA molecule on a next-generation sequencing platform at low cost, accurately distinguishing and quantifying different transcriptomic subtypes of a single gene, and better detecting the RNA 5' transcriptional regulatory region.

[0340] An eighth aspect of the invention provides a method comprising the step of measuring RNA to be tested using the sequencing method of the third aspect of the invention; or

[0341] The step includes processing the RNA to be tested using the nucleic acid composition of the fifth aspect of the present invention, the reagent combination of the sixth aspect of the present invention, or the library construction kit of the seventh aspect of the present invention;

[0342] The method includes any one of d1) to d5):

[0343] d1) Methods for detecting / locating transcription start sites at the 5' end of RNA; d2) Methods for detecting the 5' end UTR of RNA; d3) Methods for detecting the 5' end ORF of RNA; d4) Methods for distinguishing different transcriptomic subtypes of a single gene; d5) Methods for quantitatively detecting different transcriptomic subtypes.

[0344] The present invention will be further described in detail below through specific embodiments.

[0345] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0346] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0347] Experimental materials used in the examples: nuclease-free centrifuge tubes and pipette tips, Qubit fluorescence detector, total RNA from GM12878 cells, 80% ethanol, nuclease-free water, agarose, RNA Cleanup beads (BECKMAN, NC0068576), AMPure XPbeads (BECKMAN, 19800900), 5M NaOH, 1M Tris-HCl pH 7.5, 0.5M EDTA, 5'-Polyphosphatase (Lucigen, RP8092H), Quick CIP (NEB, M0525S), DNase I (Ambion, AM2222), SUPERase In (Invitrogen, AM2696), RNA 5'-Pyrophosphohydrolase (RppH, NEB, M0356), Dynabeads TM MyOne TM Streptavidin C1 beads (Invitrogen, 650001), T4 RNAligase (TAKARA, 2050A), SMARTScribe TM Reverse Transcriptase (TAKARA, 639536), 2×KAPAHiFi (Roche, KK2602), 10× ThermoPol Reaction Buffer (NEB, B9004S), Taq DNApolymerase (NEB, M0273X), dNTP (NEB, N0447V), T4 DNA ligase (NEB, M0202T), ATP (Thermol, R0440), Exol (NEB, M0293S), ExoIII (NEB, M0206L).

[0348] C1 2×B&W buffer: 10mM Tris-HCl pH 7.5, 1mM EDTA, 2M NaCl; C1 buffer A: DEPC-treated 0.1M NaOH, DEPC-treated 0.05M NaCl; C1 buffer B: DEPC-treated 0.1M NaCl; C1 hybridization buffer (10×): 100mM Tris-HCl pH 7.5, 10mM EDTA, 1M NaCl; 10×TA buffer: 330mM Tris-HCl pH 7.5, 660mM KAc, 100mM MgAc, 5mM DTT; 10× annealing buffer: 100mM Tris-HCl pH 7.5, 1M NaCl; 3×HB buffer: 30% PEG-8000, 150mM Tris-HCl pH 8.3, 30mM MgCl2, 3mM ATP, 0.15mg / mL BSA.

[0349] The sequences of primers and adapters used in the embodiments are shown in Table 1.

[0350] Table 1 Primer and adapter sequences

[0351]

[0352]

[0353] Note: r represents ribonucleotide.

[0354] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0355] Example 1: Precise detection of transcription start site and 5' UTR and ORF sequences in GM12878 cells.

[0356] This embodiment provides a method for accurately detecting the transcription start site, 5' UTR, and ORF sequences in GM12878 cells, including the following steps:

[0357] (1) Purification of 5'-capped mRNA: Take 200 ng of total RNA from cells and incubate at 37°C for 30 min according to reaction system 1 (Table 2); add reaction system 2 (Table 3), incubate at 37°C for 10 min, purify the reaction product with 1.5×RNA Cleanup beads, and elute with 40 μL of nuclease-free water.

[0358] Table 2 Reaction System 1

[0359]

[0360] Table 3 Reaction System 2

[0361]

[0362] (2) 5'-decap: Prepare the following reaction system (Table 4), incubate at 37℃ for 60 min, purify the reaction product with 1.5×RNA Cleanupbeads, and add 24 μL of nuclease-free water for elution.

[0363] Table 4. Reaction system of 5'-decap

[0364]

[0365] (3) 5' adapter ligation: Prepare the reaction system as shown in Table 5, incubate overnight at 15℃, purify the reaction product with 1.5×RNA Cleanupbeads, add 10μL of nuclease-free water to elute, and obtain product (3).

[0366] Table 5. Reaction system for 5' connector connection

[0367]

[0368] (4) Purification of 5' linker RNA:

[0369] a. Equilibrate Streptavidin C1 magnetic beads at room temperature for 30 min, mix well, and take 20 μL for later use. Rinse the magnetic beads twice with 20 μL of 1×B&W buffer, buffer A, and buffer B respectively, and store in 10 μL of 2×B&W buffer.

[0370] b. Mix 1 μL Capture Probe (100 μM) with 9 μL product (3), incubate at 80 °C for 10 min, and slowly cool to room temperature;

[0371] c. Mix the annealing product with the magnetic beads and incubate at RT and 800 rpm for 15 min. Wash twice with 1×B&W buffer and nuclease-free water, respectively.

[0372] d. Add 10 μL of nuclease-free water and incubate at 80 °C and 800 rpm for 15 min. Immediately place the mixture on a magnetic rack and collect the supernatant to obtain product (4).

[0373] (5) Reverse transcription: Add 2 μL of 20 μM RT primer 17c_mRNA and 1 μL of 10 mM dNTPs to 9 μL of product (4), incubate at 72℃ for 3 min, and then quickly place on ice for 2 min; prepare the reaction system as shown in Table 6, incubate (42℃ for 90 min, 70℃ for 15 min), and store at 4℃; purify with 1×AMPure XP magnetic beads and dissolve in 13 μL of 1×TE buffer.

[0374] Table 6 Reverse Transcription Reaction System

[0375]

[0376] (6) First PCR (PCR 1): Prepare the reaction system as shown in Table 7 and react under the following conditions: 98℃ for 3 min; 8 cycles: 95℃ for 20 sec, 62℃ for 15 sec, 72℃ for 5 min; purify the PCR product with 1×AMPure XP magnetic beads, add 35 μL of 1×TE buffer to elute, and measure the concentration with Qubit.

[0377] Table 7 Reaction system for PCR1

[0378]

[0379] (7) First cyclization reaction 1 (cyclization reaction 1): Prepare reaction system 1 as shown in Table 8, incubate at room temperature for 5 min, add 2 μL of 1M Tris-HCl pH 7 to obtain DNA from reaction 1; prepare reaction system 2 as shown in Table 9, and cycle the reaction for 54 times under the following conditions: 15℃ for 30 sec, 37℃ for 30 sec, then incubate at 60℃ for 10 min to obtain DNA from reaction 2; prepare reaction system 3 as shown in Table 10, incubate at 37℃ for 30 min, and add 2 μL of 0.5M EDTA to terminate the reaction. Purify the product with 100 μL (2.5×) magnetic beads, elute with 25 μL of 1×TE buffer, and measure the concentration using a Qubit.

[0380] Table 8 Reaction System 1

[0381]

[0382] Table 9 Reaction System 2

[0383]

[0384] Table 10 Reaction System 3

[0385]

[0386]

[0387] (8) Reverse control extension: Prepare the reaction system as shown in Table 11 and react under the following conditions: 95℃ for 2 min, 54℃ for 1.5 min, and 68℃ for 10 min.

[0388] Table 11 Reaction system for reverse control extension

[0389]

[0390] (9) End ligation: Prepare stcLFR20 / 21 (45 μM, 100 μL): Take 45 μL of 100 μM stcLFR20 and stcLFR21 primers respectively, mix with 10 μL of 10× annealing buffer, incubate at 65℃ for 5 min, and then slowly cool to room temperature to prepare the reaction system as shown in Table 12. React for 54 cycles under the following conditions: 15℃ for 30 sec, 37℃ for 30 sec. Purify the PCR product with 1×AMPure XP magnetic beads and elute with 40 μL of 1×TE buffer.

[0391] Table 12 Reaction systems with end connections

[0392]

[0393] (10) Second PCR (PCR 2): Prepare the reaction system as shown in Table 13 and react under the following conditions: 98℃ for 3 min; 10 cycles: 95℃ for 20 sec, 58℃ for 30 sec, 72℃ for 1 min. Purify the PCR product with 1×AMPure XP magnetic beads, elute with 30 μL of 1×TE buffer, and measure the concentration using a Qubit.

[0394] Table 13 Reaction system for PCR 2

[0395]

[0396] (11) Second cyclization reaction (cyclization reaction 2): Prepare reaction system 1 as shown in Table 14, incubate at room temperature for 5 min, add 2 μL of 1M Tris-HCl pH 7 to obtain DNA from reaction 1; prepare reaction system 2 as shown in Table 15, and react for 54 cycles under the following conditions: 15℃ for 30 sec, 37℃ for 30 sec, then incubate at 60℃ for 10 min to obtain DNA from reaction 1; prepare reaction system 3 as shown in Table 16, incubate at 37℃ for 30 min, and add 2 μL of 0.5M EDTA to terminate the reaction. Purify the product with 100 μL (2.5×) magnetic beads, elute with 25 μL of 1×TE buffer, and measure the concentration using a Qubit.

[0397] Table 14 Reaction System 1

[0398]

[0399] Table 15 Reaction System 2

[0400]

[0401] Table 16 Reaction System 3

[0402]

[0403] (12) Sequencing: Different sequencing strategies are selected according to the length of the PCR3 amplification product as follows: If the length of the inserted fragment is no more than 300 bp, select the PE150 sequencing scheme; if the length of the inserted fragment is greater than 400 bp, select the SE400-600 sequencing scheme.

[0404] (13) Data analysis.

[0405] Specific information on library construction and sequencing protocols is as follows: Figure 1 , Figure 2 As shown, the agarose gel electrophoresis analysis results during library construction are as follows: Figure 3 As shown.

[0406] Figure 4 The image shows an example of transcription start site and 5' region mapping results. This embodiment can accurately detect the 5' start site / UTR and part of the ORF region. Simultaneously, the expression levels of different transcriptomic isoforms of the example gene are statistically analyzed using single-molecule tags.

[0407] Example 2

[0408] This embodiment provides a precise detection method for the transcription start site, 5' UTR, and ORF sequences of GM12878 cells. The only difference from Embodiment 1 is that the 5' linker RNA purification step (4) is not required, and the product obtained in step (3) is directly used for reverse transcription.

[0409] Example 3

[0410] This embodiment provides a precise detection method for the transcription start site, 5' UTR, and ORF sequences of GM12878 cells. Based on Example 1, this method further controls the length of the extended fragment and combines it with current MGI longer read sequencing methods such as SE600 to obtain longer read libraries and sequencing results with better coverage and accuracy; or removes the controlled extension step and directly sequences the full length of the PCR 1 product using PacBio, Nanopore, or Cyclone full-length sequencing technologies.

[0411] The detection method of Example 1 was further used to analyze the 5' end of the RNA of hepatitis B virus (HBV) by means of the terminal transferase activity of reverse transcriptase. The experimental procedure of the terminal transferase activity of reverse transcriptase is roughly as follows: after extracting total RNA, reverse transcription was performed (the reverse transcription process is the same as step (5) of Example 1, the only difference being that 1 μL of 100 μM TSO primer was added to the 20 μL reaction system in Table 6, the nucleotide sequence of TSO primer is GTTCAGAGTTCTACAGTCCGACGATCrGrGrG (SEQ ID NO:12)), and then the determination and analysis were performed according to steps (6) to (13) in Example 1.

[0412] The results are as follows Figure 5 As shown, the detection method in Example 1 has a lower background and is more accurate in detecting expression levels.

[0413] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for constructing an RNA library, comprising the following steps: Screen for mRNAs with 5' end caps; The 5' end of the mRNA is ligated to the 5' RNA adapter to obtain the ligation product; The ligation product was reverse transcribed using reverse transcription primers to obtain first-strand cDNA. The first-strand cDNA was amplified by PCR using 3' end primers and RNA adapter primers to obtain an RNA library; wherein the reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT).

2. The method according to claim 1, characterized in that, The amplification binding sequence in the reverse transcription primer is at least partially identical to that of the 3' end primer; Preferably, the 3' primer includes a 5' end modification; Preferably, the 5' end modification includes at least one of phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl modification, or 3'-O-azidomethyl modification.

3. The method according to claim 1, characterized in that, The 5' RNA adapter is a single linker; Preferably, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide; Preferably, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides and deoxyribonucleotides; Preferably, the RNA adapter primer is at least partially reverse complementary to the 3' end of the 5' RNA adapter.

4. The method according to any one of claims 1 to 3, characterized in that, The single-molecule tag sequence includes a unique molecular tag sequence and a sample tag sequence; Preferably, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base; Preferably, the length of the unique molecular tag sequence is 4–20 bp; Preferably, the sample tag sequence is located at the 3' end of the unique molecular tag sequence; Preferably, the length of the sample label sequence is 4 to 20 bp.

5. The method according to claim 4, characterized in that, The mRNA is pretreated before being linked to the 5' RNA adapter at the 5' end. Preferably, the pretreatment includes 5'-cap mRNA purification and 5'-decap treatment; Preferably, the purification process of the 5'-cap mRNA includes the step of treating the mRNA with at least one of RNA polyphosphatase, exonuclease, CIP enzyme, endonuclease or RNase inhibitor; Preferably, the RNA polyphosphatase includes at least one of aluminum-inducible RNA 5' polyphosphatase, Escherichia coli RNA 5' polyphosphatase I, or Shigella RNA 5' polyphosphatase I; Preferably, the exonuclease includes at least one of 5' phosphate-dependent exonuclease, *Saccharomyces cerevisiae* XrnI ribonuclease, Exonuclease I, Exonuclease II, or Exonuclease III; Preferably, the endonuclease includes at least one of deoxyribonuclease DNase I, deoxyribonuclease DNase II, deoxyribonuclease, ribonuclease, micrococcal nuclease, endonuclease dsDNase, salt-active endonuclease SAN, or endonuclease Vvn. Preferably, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin. Preferably, the 5'-decap treatment involves treating the 5'-decap RNA with RNA 5' pyrophosphate hydrolase (RppH) to remove the 5' cap structure, leaving a single phosphate group.

6. The method according to claim 3, characterized in that, The system that links the 5' end of RNA to the 5' RNA linker includes RNA, 5' RNA linker, ligase, RNase inhibitor, and buffer. Preferably, the ligase includes at least one of E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circularization ligase, or a truncated form of T4 RNA ligase 2. Preferably, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin. Preferably, the buffer solution comprises at least one of dNTPs, enhancers, stabilizers, or metal ions; Preferably, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween; Preferably, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO; Preferably, the metal ion includes at least one of magnesium ions, manganese ions, or calcium ions; Preferably, the pH of the buffer solution is 6 to 8; Preferably, the concentration of the 3' RNA adapter in the system is 1–20 μM; Preferably, the concentration of the ligase in the system is 0.1–10 U / μL; Preferably, the concentration of the reinforcing agent in the system is 0.01 to 1 v / v%. Preferably, the concentration of the stabilizer in the system is 0.01 to 0.5 μg / μL.

7. The method according to claim 6, characterized in that, The ligation product was purified prior to reverse transcription; Preferably, the purification method includes magnetic bead purification, centrifugal column purification, or a combination thereof; Preferably, the magnetic bead purification includes the following steps: mixing the ligation product with the capture probe, reacting, mixing with magnetic beads, and performing magnetic adsorption to obtain the purified ligation product; Preferably, the nucleotide sequence of the capture probe is at least partially identical to the RNA adapter sequence; Preferably, the concentration of the capture probe in the reaction system is 5–20 μM; Preferably, the reaction conditions are: incubation at 70–90°C for 5–20 minutes, followed by slow cooling to room temperature.

8. The method according to claim 4, characterized in that, The reverse transcription system includes reverse transcriptase, reverse transcription primers, and buffer solution; Preferably, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof; Preferably, the concentration of the reverse transcriptase in the system is 0.1–10 U / μL; Preferably, the concentration of the reverse transcription primer in the system is 0.1–20 μM; Preferably, the buffer solution comprises dNTPs and metal ions, and at least one of ATP and / or GTP; Preferably, the concentration of dNTPs in the buffer solution in the system is 0.05–5 mM; Preferably, the metal ion includes at least one of magnesium ions, manganese ions, or calcium ions.

9. The method according to claim 6 or 7, characterized in that, The reaction system for PCR amplification of the first strand cDNA includes a 3' end primer, an RNA adapter sequence primer, a polymerase, and a PCR enhancer. Preferably, the concentration of the 3' end primer in the reaction system is 0.1–1 μM; Preferably, the concentration of the RNA adapter sequence primer in the reaction system is 0.1–1 μM; Preferably, the polymerase comprises at least one of T4 DNA polymerase, DNA polymerase I Klenow large fragment, T7 DNA polymerase, DNA polymerase I, KAPA HiFi DNA polymerase, or Phusion DNA polymerase; preferably, the concentration of the polymerase in the reaction system is 0.5–2 U / μL. Preferably, the PCR enhancer includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween; preferably, the concentration of the PCR enhancer in the reaction system is 0.5–2 M.

10. A nucleic acid library, constructed by the method described in any one of claims 1 to 9.

11. A sequencing method, comprising the step of sequencing the nucleic acid library of claim 10; Preferably, the sequencing method includes at least one of PacBio, Nanopore, Cyclone, or DNB sequencing.

12. The sequencing method according to claim 11, characterized in that: The sequencing method is DNB sequencing; Preferably, the DNB sequencing includes any one of a1) to a2): a1) Nucleic acid was subjected to cyclization, reverse controlled extension, end ligation, cyclization, digestion of linear single strands, purification, and rolling circle replication in sequence to obtain DNA nanospheres; a2) Reverse controlled extension, end ligation, circularization, and rolling circle replication of nucleic acids are completed in one step to obtain DNA nanospheres; Preferably, the product length obtained by the controlled extension is 300–1500 bp. Application of 13.b1)~b3) in c1)~c7): b1) The RNA library as described in any one of claims 1 to 9; b2) The nucleic acid library according to claim 10; b3) The sequencing method according to claim 11 or 12; c1) Detect / locate the 5' transcription start site of RNA; c2) Detect the 5' UTR of RNA; c3) Detect the 5' end ORF of RNA; c4) Distinguish between different transcript subtypes of a single gene; c5) Quantitative detection of different transcript subtypes; c6) gene expression regulation and function study; c7) Gene editing.

14. A nucleic acid composition comprising a 5' RNA linker and a reverse transcription primer; Preferably, the 5' RNA adapter is a universal sequence; Preferably, the nucleotide at the 5' end of the 5' RNA linker is a ribonucleotide; Preferably, the nucleotides other than the nucleotide at the 5' end of the 5' RNA linker are each independently selected from ribonucleotides and deoxyribonucleotides; Preferably, the reverse transcription primers include an amplification binding sequence, a single-molecule tag sequence, and oligo(dT); Preferably, the single-molecule tag sequence includes a unique molecular tag sequence and a sample tag sequence; Preferably, the unique molecular tag sequence is a sequence of multiple N bases, where N represents a random base; Preferably, the length of the unique molecular tag sequence is 4–20 bp; Preferably, the sample tag sequence is located at the 3' end of the unique molecular tag sequence; Preferably, the length of the sample label sequence is 4 to 20 bp.

15. A reagent combination for 5' RNA linker ligation-reverse transcription, comprising the nucleic acid composition of claim 14, a ligase, and a reverse transcriptase; Preferably, the ligase can catalyze the formation of phosphodiester bonds between the 5'-P end and the 3'-OH end of single-stranded oligonucleotides or mononucleotides, either intermolecularly or intramolecularly. Preferably, the ligase includes at least one of E. coli DNA Ligase, T4 RNA ligase 1, TS2126 RNA ligase, single-stranded DNA / RNA circularization ligase, or a truncated form of T4 RNA ligase 2. Preferably, the reverse transcriptase includes M-MLV reverse transcriptase, AMV reverse transcriptase, or a combination thereof.

16. The reagent combination according to claim 15, characterized in that: The reagent combination also includes a buffer solution; Preferably, the buffer solution comprises at least one of dNTPs, enhancers, stabilizers, metal ions, or inhibitors; Preferably, the reinforcing agent includes at least one of betaine, trehalose, glycerol, DMSO, polyethylene glycol, formamide, ammonium sulfate, tetramethylammonium chloride, gelatin, BSA, Triton X-100, or Tween; Preferably, the stabilizer includes at least one of BSA, sucrose, trehalose, polyethyleneimine, dithiothreitol, or DMSO; Preferably, the metal ion includes at least one of magnesium ions, manganese ions, or calcium ions; Preferably, the inhibitor includes an RNase inhibitor; Preferably, the RNase inhibitor includes at least one of Murine RNase inhibitor, diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, SDS, urea, diatomaceous earth, or RNasin. Preferably, the pH of the buffer solution is 6 to 8.

17. A library construction kit comprising the nucleic acid composition of claim 14 and / or the reagent combination of claim 15 or 16; Preferably, the library construction kit further includes DNB preparation reagents; Preferably, the DNB preparation reagent comprises a polynucleotide kinase; Preferably, the polynucleotide kinase is T4 polynucleotide kinase; Preferably, the DNB preparation reagent comprises substances selected from any of the following DNB preparation methods: a1) Nucleic acid was subjected to cyclization, reverse controlled extension, end ligation, cyclization, digestion of linear single strands, purification, and rolling circle replication in sequence to obtain DNA nanospheres; a2) The reverse controlled extension, end ligation, circularization reaction and rolling circle replication of nucleic acid are completed in one step to obtain DNA nanospheres; Preferably, the DNB preparation reagent includes single-tube long fragment reading primers and circularization primers; Preferably, the library construction kit further includes an exonuclease; Preferably, the exonuclease includes at least one of Exonuclease I, Exonuclease II, or Exonuclease III.

18. The library construction kit according to claim 17, characterized in that, The library construction kit also includes polymerase; Preferably, the polymerase comprises at least one of T4 DNA polymerase, DNA polymerase I Klenow large fragment, T7 DNA polymerase, DNA polymerase I, KAPA HiFi DNA polymerase, or Phusion DNA polymerase; preferably, the library construction kit comprises a buffer. Preferably, the buffer solution comprises at least one of dNTPs, metal ions, or inhibitors; Preferably, the metal ion includes at least one of magnesium ions, manganese ions, or calcium ions; Preferably, the inhibitor includes an RNase inhibitor; Preferably, the pH of the buffer solution is 6 to 8.

19. A method comprising the step of measuring the RNA to be tested using the sequencing method of claim 11 or 12; or The steps include processing the RNA to be tested using the nucleic acid composition of claim 14, the reagent combination of claim 15 or 16, or the library construction kit of claim 17 or 18; The method includes any one of d1) to d5): d1) Methods for detecting / locating the 5' transcription start site of RNA; d2) Methods for detecting the 5' end UTR of RNA; d3) Methods for detecting the 5' end ORF of RNA; d4) Methods for distinguishing different transcriptomic subtypes of a single gene; d5) Methods for quantitative detection of different transcript subtypes.

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