WO2024222158A1 - Targeted high-throughput sequencing method for detecting splicing isoform - Google Patents

Abstract

A targeted high-throughput sequencing method for detecting a splicing isoform, which method comprises a method for establishing a sequencing library for high-throughput sequencing. The method for establishing the sequencing library comprises the following steps: 1) performing reverse transcription on a sample RNA using a reverse transcription primer, adding common dNTP and 3'-modified dNTP, and reacting to obtain a first chain of cDNA; 2) connecting an oligonucleotide fragment with alkynyl modification at the 5' end with the cDNA fragment obtained in the step 1) by means of a click chemical reaction; 3) performing PCR amplification using the reaction product in the step 2) as a template; and 4) obtaining a sequencing library. A target enrichment primer can be introduced in the step of reverse transcription or PCR amplification. The reaction system of the enrichment step comprises multiple gene-specific primers, and each primer is designed on the basis of a downstream exon segment of an alternative splicing event in the transcript, to ensure that random-length fragments generated by reverse transcription cover splicing sites.

Description

A targeted high-throughput sequencing method for detecting splicing isoforms

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT international application No. PCT/CN2023/091367 filed on April 27, 2023, and this application cites the full text of the above-mentioned PCT international application.

Technical Field

The present invention relates to the field of biotechnology, and in particular to a targeted high-throughput sequencing method for detecting splicing isoforms.

Background Art

Alternative splicing (AS) refers to the process of generating different mRNA splicing isoforms from a single mRNA precursor through different splicing modes (selecting different splicing site combinations). There is a certain degree of flexibility in the process of alternative splicing, and the final mRNA structure can selectively contain some exon and/or intron fragments of the precursor RNA, thereby causing changes in the sequence composition of the protein. Therefore, alternative splicing is an important mechanism for proteome diversity.

In the process of gene expression, gene transcription first synthesizes pre-mRNA, which contains all introns and exons as well as non-coding regions at both ends (5’ and 3’ Untranslated regions). RNA splicing is an essential step for pre-mRNA to mature into mRNA. Under the action of the spliceosome, introns in pre-mRNA are removed and all exons and UTR sequences are connected. When alternative splicing occurs, the same pre-mRNA can generate two or more splice isomers. Alternative splicing can be summarized into five forms, of which the two most common ones are intron retention and exon skipping. As shown in Figure 1 below, intron retention means that introns are not removed during pre-mRNA splicing and are still retained on mRNA; exon skipping means that exons are removed by spliceosomes during pre-mRNA splicing, resulting in exon deletion in the subsequently generated mRNA.

The commercial RNA library construction kits currently available on the market mainly use different fragmentation methods to construct total mRNA sequencing libraries, and there is no targeted enrichment library construction method for splicing isoforms, especially for unknown splicing isoforms. The main reason is that the classic RNA-seq is to randomly fragment the total RNA (total RNA) or messenger RNA (messenger RNA, mRNA), and then reverse transcribe it through random primers random hexamer and/or oligo (dT) primers to form double-stranded DNA and complete the subsequent adapter connection library construction. The above method only targets the whole transcriptome, and cannot target a small part of the transcriptome of interest for analysis, and the cost is high. Since the fragment positions obtained by RNA-seq are random and usually not at the splicing intersection, extremely high sequencing depth is required to accurately detect different splicing isoforms, especially for many potential alternative splicing sites to be verified, so the cost of sequencing is extremely huge. For the currently available commercial targeted RNA sequencing kits on the market, they are all kits designed and developed for known targets, and cannot meet the requirements for target sequencing of unknown splicing isoforms. At the same time, the targeted RNA sequencing methods reported in existing literature are also difficult to apply to enterprises and non-scientific research purposes. The main reason is that it is necessary to rely on commercial companies such as Agilent Technologies and IDT to design and synthesize hybridization probes for targeted enrichment. This process is usually time-consuming and requires continuous optimization, and it is impossible to flexibly change (add, delete) the existing probes. New potential targets discovered in real time cannot be verified in a timely and efficient manner, and the cost is huge. Even for optimized and mature targeted enrichment systems, the operation cycle of library construction usually takes at least 2 days, and the hands-on steps are complicated. In addition, splicing isoform verification is required. One of the key points is to include all isoforms as much as possible, but all current methods may not be perfect for this. Another major factor is that the existing method (Agilent) can only analyze known splice isoforms: the need for primer design, the possibility of competition between amplification primers of different splice isoforms, even in a single droplet with highly diluted template. The classic method for quantitative analysis of low-throughput splice isoforms is qRT-PCR, but the different amplification efficiencies between different splice isoforms will cause systematic deviations in the fluorescence signal, and only relative quantification can be achieved even through cumbersome calibration steps; and when detecting low-abundance splice isoforms, the requirements for primer design, experimental operation and equipment are high, and the quantitative results are difficult to repeat.

Therefore, it is particularly important and urgent to develop a more efficient and accurate RNA multiple targeted enrichment library construction method.

Summary of the invention

In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide a targeted high-throughput sequencing method for detecting splicing isoforms.

In order to achieve the above objectives, the present disclosure adopts the following specific technical solutions:

In one aspect, the present disclosure provides a method for establishing a sequencing library for high-throughput sequencing, comprising the following steps:

(1) reverse transcription of sample RNA using a reverse transcription primer, adding common dNTP and 3'-modified dNTP to obtain a first cDNA chain, wherein the 3'-modified dNTP is selected from one or more of the following: AzNTP, AmNTP, propargyl-NTP, HalNTP;

(2) connecting the oligonucleotide fragment with an alkyne modification at the 5′ end to the cDNA fragment obtained in step (1) by a click chemistry reaction, wherein the oligonucleotide fragment comprises a random sequence and a complementary segment sequence of universal sequencing primer 1 (seq1);

(3) using the reaction product of step (2) as a template, performing PCR amplification;

(4) Obtain a sequencing library.

In another aspect, the present disclosure provides a high-throughput sequencing library constructed according to the above method.

In another aspect, the present disclosure provides a targeted high-throughput sequencing method for detecting splicing isoforms, comprising the following steps:

(1) extracting RNA from target cells and constructing a sequencing library according to the above method;

(2) Based on the above sequencing library, high-throughput sequencing is performed to obtain sequencing information of the target gene in the cell sample.

In another aspect, the present disclosure provides a method for establishing a sequencing library for high-throughput sequencing, a use of the high-throughput sequencing library and/or the targeted high-throughput sequencing method in the evaluation of off-target events;

Preferably, the off-target event assessment includes the following:

(1) Accurately determine the genomic location where off-target trans-splicing occurs in trans-splicing factors;

(2) Quantitative analysis of on-target trans-splicing and off-target trans-splicing.

The beneficial effects achieved by the present disclosure are at least as follows:

(1) Realize the analysis of multiple targeted splicing isoforms. The throughput of traditional qRT-PCR methods is limited by the number of fluorescence in the detection instrument, bleed-through between fluorescence, and mutual interference between primers in the reaction system. The throughput of the commonly used qRT-PCR is quadruple, while the HTAS (High-throughput Targeted Alternative Splicing) analysis platform disclosed in the present invention can simultaneously analyze 5-100 (or more) pre-mRNA splicing events;

(2) Accurate quantification of splicing isoforms: High-throughput sequencing performs quantitative analysis of splicing isoforms at the single-molecule level (digital signal), while traditional qRT-PCR quantification relies on fluorescence intensity (analog signal). Analog signals only support relative quantification. Digital signals can achieve absolute quantification of sample splicing isoforms. In addition, the unique advantage of HTAS is that the addition of a certain proportion of 3' modified dNTPs during reverse transcription can randomly terminate the extension of the first-chain cDNA, so the same type of splicing isoforms can generate cDNA products of different lengths, and the amplification efficiency is no longer affected by the different lengths of splicing isoform PCR products during library construction. Finally, the introduction of random sequences (N5; up to 12-16 nucleotides) in the design of the 5' linker can remove systematic bias (PCR skewing) in the PCR amplification process; mixed sample experiments have shown that the correlation between the actual detection value of HTAS and the theoretical value of mixed samples is r>0.99;

(2) Detection sensitivity: The minimum intron-retained (IR) splicing isoform ratio detected in the embodiment is 0.1%. With the increase of sequencing depth, the detection sensitivity of IR is expected to reach 1/10 ⁵ or lower, which is much higher than that of traditional RNA-seq, especially for low-abundance transcripts;

(4) Unknown splicing isoforms can be detected: Due to the diversity of tissues and cells, a large number of splicing isoforms in the current detection system have not been annotated in the transcriptome. Traditional qRT-PCR schemes are mainly designed for known splicing isoforms, while the HTAS platform can detect both known and unknown splicing isoforms, eliminating the interference of missing annotations in isoform analysis and improving the specificity of quantitative analysis;

(5) Realize the evaluation of trans-splicing off-target events: Trans-splicing is a new technology for the targeted editing of mRNA, but the off-target phenomenon is one of the main bottlenecks in its clinical application. Since off-target events are expected to occur in any pre-mRNA in the transcriptome, there is currently no method to systematically evaluate the off-target phenomenon. HTAS only needs to know the sequence of the trans-splicing molecule (Pre-mRNA Trans-splicing Molecule) to simultaneously perform qualitative and quantitative analysis of on-target trans-splicing and off-target trans-splicing;

(6) Simple operation and low cost: No complicated library construction and enrichment steps are required, and the entire library construction time is about 4-5 hours. The cost per sample is significantly lower than existing products or methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the principle of splicing isomerization.

Figure 2 is a schematic diagram of the high-throughput sequencing library construction (Scheme 1).

Figure 3 is a schematic diagram of the high-throughput sequencing library construction (Scheme 2).

FIG4 is a schematic diagram of the construction principle of a high-throughput sequencing library (Scheme 3).

FIG5 is a schematic diagram showing the correlation between the actual detection values of splicing isoform IVT products with different mixing ratios and the theoretical values of mixed samples.

DETAILED DESCRIPTION

I. Definitions

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meanings as those commonly understood by those skilled in the art to which the present disclosure belongs. The terms used in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

The terms "include", "comprises", "comprising", and any variations thereof, of the present disclosure are intended to cover a non-exclusive inclusion. For example, A process, method, apparatus, product, or device that includes a series of steps is not limited to the listed steps or modules, but may optionally include unlisted steps, or may optionally include other steps inherent to these processes, methods, products, or devices.

The "plurality" mentioned in the present disclosure refers to two or more. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the objects associated before and after are in an "or" relationship. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below. As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

With respect to polypeptide sequences, the phrase "substantially identical" is understood to mean a sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term is understood to mean a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

The term "Oligo(dT) primer" used in the present disclosure refers to a repetitive oligonucleotide sequence composed of about 12-25 polythymine Ts, which can specifically anneal to the ploy(A) tail of eukaryotic mRNA, and is therefore not suitable for RNA lacking a ploy(A) tail structure, such as prokaryotic RNA or miRNA, nor for degraded RNA, such as RNA in FFPE samples. Oligo(dT)n VN is composed of a fixed and specific sequence + (T)20 or so + degenerate base VN. The fixed and specific sequence exists to facilitate the design of universal PCR downstream primers. The VN in the Oligo(dT)n VN sequence refers to the presence of an anchor base at the 3' end. ("V" represents dATP, dGTP or dCTP; "N" represents any one of dATP, dTTP, dGTP, and dCTP). The function of the anchor base is to be able to specifically bind to the 5' end of Poly(A) to prevent excessive T bases from being reverse transcribed.

The term "click chemistry" as used in the present disclosure is well known in the art and generally refers to a fast reaction that is easy to purify and region-specific. Click chemistry is a class of reactions that allow a selected substrate to be connected to a specific molecule. Click chemistry is not a single specific reaction, but describes a way of producing products according to examples in nature, which also produces substances by connecting small modular units. In many applications, click reactions connect biomolecules and reporter molecules. Click chemistry is not limited to biological conditions: the concept of "click" reactions has been used in pharmacology and various biomimetic applications. However, the application is particularly useful in the detection, localization and identification of biomolecules. A typical click reaction is the classic click reaction is the reaction of copper-catalyzed azides and alkynes to form a 5-membered heteroatom ring: Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

The terms "contacting," "adding," "reacting," "treating," and the like as used in this disclosure mean contacting one reactant, reagent, solvent, catalyst, reactive group, etc. with another reactant, reagent, solvent, catalyst, reactive group, etc. The reactants, reagents, solvents, catalysts, reactive groups, etc. may be added individually, simultaneously, or separately and may be added in any order that achieves the desired result. The reactants, the reagents, the solvents, the catalysts, the reactive groups, etc. may be added with or without heating or cooling equipment and may optionally be added under an inert atmosphere.

The term "complementary" used in this disclosure refers to the broad concept of sequence complementarity between regions of two polynucleotide chains or between two nucleotides by base pairing. It is known that adenine nucleotides can form specific hydrogen bonds ("base pairing") with thymine or uracil nucleotides. Similarly, it is known that cytosine nucleotides can base pair with guanine nucleotides.

As used in this disclosure, the term "library" when used with respect to nucleic acids is intended to mean a collection of nucleic acids having different chemical compositions (e.g., different sequences, different lengths, etc.). Typically, the nucleic acids in a library will be different species having common features or properties of a genus or class, but differing to some extent in other respects. For example, a library may contain nucleic acid species that differ in nucleotide sequence but are similar in having a sugar-phosphate backbone. Libraries may be created using techniques known in the art. The examples exemplified herein are The nucleic acid may include nucleic acids obtained from any source, including, for example, a genome (e.g., a human genome) or a mixture of genomes digested. In another example, the nucleic acid may be those obtained from a metagenomic study of a particular environment or ecosystem. The term also includes artificially created nucleic acid libraries, such as DNA libraries.

The terms "random primer" or "random hexamer primer" or "Random hexamer" or "Random hexamer primer" as used in this disclosure are well known in the art and generally refer to short oligodeoxyribonucleotides (d(N)6) of random sequence that anneal to random complementary sites on the target DNA or RNA and are used as primers for DNA synthesis by DNA polymerase or reverse transcriptase.

The term "AzNTP (3'-azido-2',3'dNTP)" as used in the present disclosure is an azide deoxynucleotide, wherein the base is selected from adenine, guanine, cytosine and thymine.

The term "Add on PCR" used in the present disclosure means that the primers involved in PCR have some other sequences in addition to the sequences complementary to the template. These other sequences do not participate in this round of PCR reaction, but the generated PCR products can serve as templates for the next PCR reaction because of the additional sequences.

II. Detailed description of specific implementation methods

In some embodiments, the present disclosure provides a method for establishing a sequencing library for high-throughput sequencing, comprising the following steps:

(1) reverse transcription of sample RNA using a reverse transcription primer, adding common dNTP and 3'-modified dNTP to obtain a first cDNA chain, wherein the 3'-modified dNTP is selected from one or more of the following: AzNTP, AmNTP, propargyl-NTP, HalNTP;

(2) connecting the oligonucleotide fragment with an alkyne modification at the 5′ end to the cDNA fragment obtained in step (1) by a click chemistry reaction, wherein the oligonucleotide fragment comprises a random sequence and a complementary segment sequence of universal sequencing primer 1 (seq1);

(3) using the reaction product of step (2) as a template, performing PCR amplification;

(4) Obtain a sequencing library.

In some embodiments, the click chemistry reaction refers to a CuAAC click reaction, i.e., a copper ion-catalyzed azide-alkyne cycloaddition reaction.

In some embodiments, the reverse transcription PCR reaction in step (1) includes five reaction stages: the specific reaction conditions are 25°C for 10 min, 37°C for 10 min, 50°C for 45 min, 85°C for 2 min, and 12°C for maintenance.

In some embodiments, the enzymes used in the reverse transcription PCR reaction in step (1) include HiScript III Reverse Transcriptase (R302-01, Nanjing Novogene Biotechnology Co., Ltd.), SuperScript ^TM III Reverse Transcriptase (18080093, ThermoFisher SCIENTIFIC), HiFi II M-MLV (H-) Reverse Transcriptase (CW0743, Kangwei Century Biotechnology Co., Ltd.), Reverse Transcriptase [M-MLV, RNaseH-] (AE101-02, Beijing Quanshijin Biotechnology Co., Ltd.), MutiScript II Reverse Transcriptase (MD311, Feipeng Biotechnology Co., Ltd.).

In some embodiments, the reverse transcription primer described in step (1) is a random primer or a gene-specific primer group 1 designed based on the downstream exon of the retained intron of the targeted gene, and the 5' end of the primer in the gene-specific primer group 1 carries a universal sequencing primer 2 sequence (seq2).

In some embodiments, the click chemistry reaction system in step (2) further includes vitamin C, a copper (II)-TBTA composition, and DMSO.

In some embodiments, the PCR amplification reaction in step (3) includes four reaction stages: the first PCR amplification reaction is 1 cycle, and the specific reaction conditions are 94°C for 1 min, 60°C for 30 s, and 68°C for 10 min; the second PCR amplification reaction includes 12 cycles, and the specific reaction conditions are 94°C for 30 s, 60°C for 30 s, and 68°C for 2 min; the third PCR amplification reaction includes 1 cycle, and the specific reaction conditions are 68°C for 5 min; the fourth PCR amplification reaction includes 1 cycle, and the specific reaction conditions are 12°C.

In some embodiments, the PCR reaction system in step (3) further includes PCR Buffer solutions such as MgCl ₂ , DMSO, Tris-HCl, EDTA, NaCl, and KCl.

In some embodiments, the enzyme used in the PCR reaction in step (3) is Taq DNA polymerase.

In some embodiments, the PCR amplification primers used in step (3) include universal sequencing primer 1 and gene-specific primer group 2, wherein the 5' end of the primer in the gene-specific primer group 2 carries the universal sequencing primer 2 sequence.

In some embodiments, both gene-specific primer groups 1 and 2 are designed based on exons downstream of alternative splicing events of retained introns of specific targeted genes, wherein the targeting site of gene-specific primer 2 is shifted 5-100 bases upstream of the site of gene-specific primer 1.

In some embodiments, the targeting site of gene-specific primer 2 is shifted upstream by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 bases than the site of gene-specific primer 1.

In some embodiments, the targeting site of gene-specific primer group 2 is shifted 20-50 bases upstream of the position of gene-specific primer group 1.

In some embodiments, the number of target genes targeted by the gene-specific primer group 1 and the gene-specific primer group 2 respectively is greater than or equal to 1.

In some embodiments, both the gene-specific primer groups 1 and 2 are designed based on the exons downstream of the retained introns of the specific targeted gene.

In some embodiments, the targeting positions of the gene-specific primer groups 1 and 2 may partially overlap but not completely coincide.

In some embodiments, the molar concentration ratio of common dNTPs and 3'-modified dNTPs added in step (1) is 1:1-1:100, and the molar concentration ratio can be 1:100, 1:90, 1:80, 1:75, 1:70, 1:65, 1:60, 1:55, 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11 or 1:10; in a preferred embodiment, the molar concentration ratio of common dNTPs and 3'-modified dNTPs added in step (1) is 1:50.

In a preferred embodiment, the molar concentration ratio of common dNTP and 3'-modified dNTP added in step (1) is 1:20.

In some embodiments, the random sequence in step (2) comprises 4-16 nucleotides.

In some embodiments, the random sequence in step (2) comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides.

In some embodiments, the random sequence in step (2) comprises 5 nucleotides.

In some embodiments, the PCR amplification of step (3) includes the step of performing PCR amplification on the reaction product of step (2) using universal sequencing primer 1 and gene-specific primer group 2, and then adding a connector structure to the amplified product, wherein the connector structure includes a P5/P7 connector and a nucleic acid barcode, and the nucleic acid barcode is connected to the primer P5 and/or P7 connector end.

In some embodiments, step (3) is: using the reaction product of step (2) as a template, adding P5 and P7 PCR reaction was performed on the first.

In some embodiments, the P5 linker primer is shown as SEQ ID NO: 54.

In some embodiments, the P7 linker primer is as shown in SEQ ID NO: 55-57.

In some embodiments, the nucleic acid barcode is attached to a single end of the primer P7 adapter.

In some embodiments, the nucleic acid barcode is attached to both ends of primer P5 and P7 adapter.

In some embodiments, the nucleic acid barcode is divided into nucleic acid barcode 5 and nucleic acid barcode 7.

In some embodiments, the nucleotide sequence of the nucleic acid barcode 5 is selected from the sequence shown in any one of SEQ ID NO.10-35.

In some embodiments, the nucleotide sequence of the nucleic acid barcode 7 is selected from the sequence shown in any one of SEQ ID NO.36-53.

In some embodiments, the PCR amplification reaction in step (3) includes four reaction stages: the first PCR amplification reaction is 1 cycle, and the specific reaction conditions are 94°C for 30 seconds; the second PCR amplification reaction includes 18 cycles, and the specific reaction conditions are 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 30 seconds; the third PCR amplification reaction includes 1 cycle, and the specific reaction conditions are 72°C for 5 minutes; the fourth PCR amplification reaction includes 1 cycle, and the specific reaction conditions are 12°C.

In some embodiments, the PCR reaction system in step (3) further includes PCR Buffer solutions such as MgCl ₂ , DMSO, Tris-HCl, EDTA, NaCl, and KCl.

In some embodiments, the enzyme used in the PCR reaction in step (3) is Taq DNA polymerase.

In some embodiments, the universal sequencing primer 1 sequence is selected from any one of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 6.

In some embodiments, the complementary segment sequence of the universal sequencing primer 1 is shown as SEQ ID NO:4.

In some embodiments, the universal sequencing primer 2 sequence (seq2) is selected from any one of SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, the present disclosure provides a high-throughput sequencing library constructed according to the above method.

In some embodiments, the present disclosure provides a targeted high-throughput sequencing method for detecting splicing isoforms, comprising the following steps:

(1) extracting sample RNA and constructing a sequencing library according to the above method;

(2) Based on the above sequencing library, high-throughput sequencing is performed to obtain sequencing information of the target gene in the cell sample.

In some embodiments, the sample is a tissue, cell, and/or body fluid sample.

In some embodiments, the body fluid sample includes one or more of blood, saliva, urine, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bile, and pleural effusion.

In some embodiments, the sample is a cell sample.

In some embodiments, the cell samples include, but are not limited to, MCF10A, MCF7, HeLa, HEK293T, and/or MDA-MB-231.

In some embodiments, the target genes include but are not limited to ATP13A1, CXXC1, ECHDC2, FGFRL1, HMGN3, KLHL17, NAXD, LZTR1, SELENBP1, JMJD8, PSMB1, HIGD2A, HNRNPAB, SMARCC1, ATP5IF1, HIGD2B, RPS21, UQCC5, NFATC3, PCNP and/or OSGEP.

In some embodiments, the present disclosure provides a method for establishing a sequencing library for high-throughput sequencing, the use of the high-throughput sequencing library and/or the targeted high-throughput sequencing method in the evaluation of off-target events.

In some embodiments, the off-target event assessment includes the following:

(1) Accurately determine the genomic location where off-target trans-splicing occurs in trans-splicing factors;

(2) Quantitative analysis of on-target trans-splicing and off-target trans-splicing.

In some embodiments, the high-throughput sequencing method can be used to detect any form of alternative splicing and any combination of transcripts in the transcriptome.

In some embodiments, the construction process of the sequencing library for high-throughput sequencing is as follows, and its principle diagram is shown in FIG2 :

1) Reverse transcription: The first strand of cDNA is synthesized by reverse transcription using RNA as a template, using a random hexamer primer as a reverse transcription primer, and adding ordinary dNTP and a certain concentration of 3' modified dNTP for reverse transcription reaction, wherein the 3' modified dNTP is AzNTP (3'-azido-2', 3'dNTP), which can prevent the addition of the next dNTP from binding during chain extension, and the 3' modified group should be used for chemical connection with the downstream linker. Random Hexamer is a single-stranded DNA with a random sequence of 6 bases, and the characteristics of the random sequence can help it randomly bind to different fragments of RNA. During the reverse transcription process, under the action of reverse transcriptase, dNTPs are added to the 3' end of the primer using RNA as a template to synthesize single-stranded cDNA. When the 3' modified dNTP replaces dNTPs and is added to the cDNA single strand, the chain extension is terminated and the synthesis of the first strand of cDNA is completed.

2) Click chemistry connection: The cDNA obtained above is subjected to a click chemistry reaction (i.e., CuAAC click reaction—monovalent copper ion-catalyzed azide-alkyne cycloaddition reaction) with an oligonucleotide modified with an alkyne group at the 5' end. The oligonucleotide adapter sequence contains a complementary segment sequence of 5'N5+NGS universal sequencing primer (seq1), which serves as a PCR primer binding site in the subsequent sequencing library construction. N5 is a sequence composed of 5 random bases, which is used to ensure the molecular diversity of the sequencing library and to remove systematic biases and sequencing errors in the PCR amplification process in the result analysis. The length of the random base sequence can be adjusted to N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, and N16. Where N is selected from any base in ATCG.

3) Targeted enrichment: Using the click chemistry reaction product as a template, the target gene fragment is targeted and enriched through PCR reaction. The primers in the PCR system include a universal sequencing primer 1 (seq1, whose 3' end sequence is complementary to the 3' end of the above-mentioned alkyne primer) and a gene-specific primer group. The primer group is designed based on the exons downstream of the retained introns of each targeted gene, and a universal sequencing primer 2 sequence (seq2) is added to the 5' end of the primer. The CLICK product contains an azide-alkyne linker, so the DNA polymerase needs to use Taq or other polymerases that can amplify this template.

4) Sequencing adapter connection and enrichment: Using the above enriched fragments as templates, PCR reaction is performed to add complete P5 and P7 adapters to the enriched fragments for subsequent NGS sequencing. A nucleic acid barcode (sample barcode) is added. Nucleic acid barcodes can achieve mixed samples during high-throughput sequencing, improve detection throughput, and reduce instrument errors during sample sequencing.

In some embodiments, the reverse transcription primer in the high-throughput sequencing library construction process is a gene-specific primer group 1 designed based on the downstream exon of the retained intron of the targeted gene, and a universal sequencing primer sequence (seq2) is added to the 5' end of the specific primer; in some embodiments, in the actual construction process, the sequencing adapter connection and enrichment can be directly performed after the click chemistry connection product is purified; in some embodiments, the construction process of the sequencing library for high-throughput sequencing is as follows, and its schematic diagram is shown in Figure 3:

1) Reverse transcription: RNA is used as a template to synthesize the first strand of cDNA by reverse transcription, and a gene-specific primer group 1 designed based on the downstream exons of the retained introns of the targeted gene is used as a reverse transcription primer, and a universal sequencing primer sequence (seq2) is added to the 5' end, and ordinary dNTP and a certain concentration of 3' modified dNTP are added to perform a reverse transcription reaction, wherein the 3' modified dNTP is AzNTP (3'-azido-2', 3'dNTP), which can prevent the addition of the next dNTP from binding during chain extension, and at the same time The 3' modification group should be available for chemical connection to the downstream linker. During reverse transcription, under the action of reverse transcriptase, dNTPs are added to the 3' end of the primer using RNA as a template to synthesize single-stranded cDNA. When the 3' modified dNTP replaces dNTPs and is added to the cDNA single strand, the chain extension is terminated and the synthesis of the first strand of cDNA is completed.

2) Click chemistry connection: The cDNA obtained above is subjected to a click chemistry reaction (i.e., CuAAC click reaction—monovalent copper ion-catalyzed azide-alkyne cycloaddition reaction) with an oligonucleotide modified with an alkyne group at the 5’ end. The oligonucleotide adapter sequence contains a complementary segment sequence of 5’N5+NGS universal sequencing primer (seq1), which serves as a PCR primer binding site in the subsequent sequencing library construction. N5 is a sequence composed of 5 random bases, which is used to ensure the molecular diversity of the sequencing library and to remove systematic deviations and sequencing errors in the PCR amplification process in the result analysis. The length of the random base sequence can be adjusted to N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, and N16.

3) Sequencing adapter connection and enrichment: Using the click chemistry reaction product as a template, add complete P5 and P7 adapters for subsequent PCR amplification. The P5 adapter can complement the Seq 1 complementary sequence in the click chemistry reaction product, and the P7 adapter can complement the Seq 2 complementary sequence generated during the PCR reaction. And add a nucleic acid barcode (sample barcode). Nucleic acid barcodes can achieve mixed samples during high-throughput sequencing, improve detection throughput, and reduce instrument errors during sample sequencing. After several rounds of PCR reactions, the P5 and P7 adapters are connected and the fragments to be sequenced are enriched for subsequent NGS sequencing. The CLICK product contains an azide-alkyne linker, so the DNA polymerase needs to use Taq or other polymerases that can amplify this template.

In some embodiments, the reverse transcription primer used in the high-throughput sequencing library construction process is a gene-specific primer group 1 designed based on the downstream exons of the retained introns of the targeted gene; in some embodiments, a specific gene PCR is performed after the click chemistry ligation reaction to remove the influence of ribosomal RNA in the template on the sequencing data; in some embodiments, the construction process of the sequencing library for high-throughput sequencing is as follows, and its principle diagram is shown in Figure 4:

1) Reverse transcription: The first strand of cDNA is synthesized by reverse transcription using RNA as a template, using a gene-specific primer group 1 designed based on the downstream exons of the retained introns of the targeted gene as a reverse transcription primer, and adding a universal sequencing primer sequence (seq2) at the 5' end, adding ordinary dNTP and a certain concentration of 3' modified dNTP for reverse transcription reaction, wherein the 3' modified dNTP is AzNTP (3'-azido-2', 3'dNTP), which can prevent the addition of the next dNTP from binding during chain extension, and the 3' modified group should be used for chemical connection of the downstream linker. During the reverse transcription process, under the action of reverse transcriptase, dNTPs are added to the 3' end of the primer using RNA as a template to synthesize single-stranded cDNA. When the 3' modified dNTP replaces the dNTPs and is added to the cDNA single strand, the chain extension is terminated, and the synthesis of the first strand of cDNA is completed.

2) Click chemistry connection: The cDNA obtained above is subjected to a click chemistry reaction (i.e., CuAAC click reaction—monovalent copper ion-catalyzed azide-alkyne cycloaddition reaction) with an oligonucleotide modified with an alkyne group at the 5’ end. The oligonucleotide adapter sequence contains a complementary segment sequence of 5’N5+NGS universal sequencing primer (seq1), which serves as a PCR primer binding site in the subsequent sequencing library construction. N5 is a sequence composed of 5 random bases, which is used to ensure the molecular diversity of the sequencing library and to remove systematic deviations and sequencing errors in the PCR amplification process in the result analysis. The length of the random base sequence can be adjusted to N4, N5, N6, N7, N8, N9, N10, N11, N12, N13, N14, N15, and N16.

3) Targeted enrichment: Using the click chemistry reaction product as a template, the target gene fragment is targeted and enriched through PCR reaction. The primers in the PCR system include a universal sequencing primer 1 (seq1, whose 3' end sequence is complementary to the 3' end of the above-mentioned alkyne primer) and a gene-specific primer group 2. Primer group 2 is designed based on the exons downstream of the retained introns of each targeted gene, but should be smaller than the target Move 5-100 bases upstream of the reverse transcription primer group 1 position, and even partially overlap, but not completely match, and add the universal sequencing primer 2 sequence (seq2) to the 5' end of the primer. The CLICK product contains an azide-alkyne linker, so the DNA polymerase needs to use Taq or other polymerases that can amplify this template.

4) Sequencing adapter connection and enrichment: Using the above enriched fragments as templates, PCR reaction is performed to add complete P5 and P7 adapters to the enriched fragments for subsequent NGS sequencing. A nucleic acid barcode (sample barcode) is added. Nucleic acid barcodes can achieve mixed samples during high-throughput sequencing, improve detection throughput, and reduce instrument errors during sample sequencing.

In some embodiments, the fragments in the constructed high-throughput sequencing library all include the following elements: P5 sequence, sample tag 5, universal sequencing primer 1 (seq1), inserted DNA fragment, universal sequencing primer 2 (seq2), sample tag 7, and P7 adapter.

In some embodiments, the nucleotide sequences of the P5 sequence and P7 are shown as SEQ ID NO:1 (AATGATACGGCGACCACCGAGATCTACAC) and SEQ ID NO:2 (CAAGCAGAAGACGGCATACGAGAT), respectively.

In some embodiments, the universal sequencing primer includes two types, PE adapter and Nextera adapter.

In some embodiments, the universal sequencing primers 1 and 2 are selected from any combination of the sequences in Table 1 below.

Table 1 Sequence information of universal sequencing primers

In some embodiments, the nucleotide sequence of the oligonucleotide Hex_N5_Seq1rc is as shown in SEQ ID NO:9 (NNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT).

In some embodiments, the sample tag 5 and the sample tag 7 are selected from any combination of the sequences in Table 2 below.

Table 2 Sequence information

In some embodiments, the sequence information of the P5 and P7 linkers of the complete sequence is shown in Table 3.

Table 3 Sequence information

Example

The technical solution of the present disclosure is further described below by way of specific implementation. The embodiments described above are only to help understand the present disclosure and should not be considered as specific limitations of the present disclosure.

Example 1: Construction of a targeted high-throughput sequencing platform for detecting splicing isoforms

1. RNA extraction

Use a commercial kit to extract RNA from target cells, such as the FineProtect Universal RNA Extraction Kit from Jifan Biotechnology (Beijing) Co., Ltd. The quality of RNA extraction must be guaranteed during the extraction process for subsequent experiments.

2. RNA Quantification

RNA is quantified using a commercial kit, and the RNA concentration is specifically detected using fluorescent dye technology, such as Invitrogen's Qubit ^™ RNA High Sensitivity (HS) Quantification Kit.

3. Reverse transcription

According to the quantitative concentration determined above, select RNA of the same mass as the template for synthesizing the first chain of cDNA, use random hexamer as the reverse transcription primer, and add ordinary dNTP and 3' modified dNTP at a molar concentration ratio of 1/20 to the reverse transcription system for reverse transcription reaction.

The components of the reverse transcription reaction system are shown in Table 4, and the reverse transcription reaction conditions are shown in Table 5.

Table 4: Transcription reaction system (20 μL/reaction)

Table 5: Reverse transcription reaction conditions

4. cDNA Purification

After the First-strand cDNA synthesis is completed, the product is purified using a commercial kit. For example, Zymo The purified product was eluted with pure water to 10 μL.

5. Click ligation

The purified cDNA and oligonucleotides containing alkyne modification at the 5' end were subjected to a click reaction (i.e., CuAAC click reaction - monovalent copper ion-catalyzed azide-alkyne cycloaddition reaction) in the presence of the catalyst Copper(II)-TBTA complex at room temperature for 1 hour.

The components of the click chemistry reaction system are shown in Table 6, and the nucleic acid sequence of oligonucleotides is shown in SEQ ID NO:9.

Table 6: Click chemistry reaction system (37.8 μL/reaction)

6. Click ligation product purification

The click chemistry reaction product is purified using a commercial kit, such as the DNA Clean&Concentrator-5 kit from Zymo. The purified product is eluted with pure water to 10 μL.

7. Targeted Enrichment

The purified product was used as a template, and the primers designed for the downstream exons of each retained intron of the targeted gene were used as the targeted gene-specific primer combination and a universal sequencing primer (PE1_p26) for PCR reaction.

The components of the PCR reaction system are shown in Table 7, the preparation of the primer combination mixture is shown in Table 8, the nucleic acid sequence of the PE1_p26 primer is shown in SEQ ID NO: 6, and the qPCR reaction conditions are shown in Table 9.

Table 7: PCR reaction system (25 μL/reaction)

Table 8: Primer combination mixture composition table

Table 9: PCR reaction conditions

8. Purification of targeted enrichment products

The targeted enrichment PCR product was purified using a commercial kit, such as the DNA Clean & Concentrator-5 kit from Zymo. The purified product was eluted with pure water to 10 μL.

9. Sequencing adapter ligation and enrichment

The above-obtained targeted enriched fragments are used as templates and the complete P5 and P7 adapters are used as primers to perform PCR reactions for subsequent NGS sequencing. The P5 adapter can complement the sequence containing the Seq 1 complementary segment sequence in the click reaction product, and the P7 adapter can complement the sequence containing the Seq 2 complementary sequence generated during the PCR reaction. A nucleic acid barcode (sample barcode) is added to a single Add-on PCR primer or two Add-on PCR primers to achieve mixed samples during high-throughput sequencing.

The components of the PCR reaction system are shown in Table 10, the nucleic acid sequences of the complete sequence of the P5 and P7 adapter primers are shown in Table 3, and the qPCR reaction conditions are shown in Table 11.

Table 10: PCR reaction system (25 μL/reaction)

Table 11: PCR reaction conditions

10. High-throughput sequencing and result analysis

Based on the sequencing library amplified in the above steps, high-throughput sequencing is performed to obtain sequencing information of the target gene in the cell sample, including but not limited to analysis of multiple targeted splicing isoforms, precise quantification of splicing isoforms, evaluation of unknown splicing isoforms and trans-splicing off-target events, etc.

Example 2: Detection of splicing isoforms in MCF10A, MCF7, and MDA-MB-231 cell lines

The high-throughput sequencing platform described in Example 1 was used to detect splicing isoforms in MCF10A, MCF7, and MDA-MB-231 cell lines.

Wherein, steps 1-6 are the same as those in Example 1.

In the targeted enrichment process of step 7, primers designed for the downstream exons of the retained introns of the ATP13A1 gene, CXXC1 gene, ECHDC2 gene, FGFRL1 gene, HMGN3 gene, KLHL17 gene, and OSGEP gene were used as targeted enrichment primer combinations and universal sequencing primer PE1_p26 for PCR reaction. The components of the primer combination mixture are shown in Table 12, and the primer sequences of each targeted gene are shown in Table 13.

Table 12: Primer combination mixture composition table

Table 13: Nucleic acid sequences of primers

Steps 8-10 are the same as those in Example 1.

The high-throughput sequencing results showed that the target genes of MCF10A, MCF7, and MDA-MB-231 cell lines all had normal spliceosomes and splice isoforms (intron retention), and the ratio of splice isoforms in the targeted genes was successfully quantitatively detected. The specific test results are shown in Table 14. The above data show that the targeted high-throughput sequencing platform constructed in the present disclosure can effectively realize the detection of splice isoforms.

Table 14: Detection results of target genes in MCF10A, MCF7, and MDA-MB-231 cell lines

Example 3: In-vitro Transcription (IVT) RNA simulation detection of different ratios of splicing isoforms

Prepare an in vitro RNA synthesis sample (IVT product), including two splice isoform sequences, namely, a normal splice isoform and a splice isoform (intron retention). Mix the splice isoforms of the IVT product in different proportions, and use the targeted high The throughput sequencing platform detects and calculates the correlation between the actual detection value of the mixed sample and the theoretical value of the mixed sample. The specific experimental details are as follows:

Step 1: Obtain target RNA synthesis sample by in vitro transcription

The synthesized normal spliceosome and splice isoform (intron retention) DNA is subjected to in vitro transcription reaction using commercial kits, such as Novazonic T7 High Yield RNA Transcription Kit (TR101).

The in vitro transcription reaction system is shown in Table 15.

Table 15: In vitro transcription system (20 μL system)

After incubating the above system at 37°C for 2 hours, add 1 μL DNase I and continue incubating at 37°C for 15 minutes to remove the DNA template.

Step 2: RNA recovery and purification

The RNA synthesized by in vitro transcription was recovered by phenol-ethanol precipitation. The final precipitated RNA product was dissolved in double distilled water, the RNA concentration was determined by Nano-Drop, and the RNA was diluted to 2 μM, 0.2 μM, 0.02 μM, and 0.002 μM for later use.

Step 3: Mixing different ratios of splice isoform IVT products

The IVT products were mixed in a 1:1 volume ratio as shown in Table 16.

Table 16: IVT product mix

Step 4: Reverse transcription

The mixed IVT products were diluted 100 times and used as templates for synthesizing the first chain of cDNA. The reverse primer on the downstream exon of the target intron was used as the reverse transcription primer. Ordinary dNTP and 3' modified dNTP at a molar concentration of 1/15 were added to the reverse transcription system for reverse transcription reaction.

The components of the reverse transcription reaction system are shown in Table 17, and the reverse transcription reaction conditions are shown in Table 18.

Table 17: Transcription reaction system (20 μL/reaction)

Table 18: Reverse transcription reaction conditions

Among them, steps 5-6 are performed the same as steps 4-6 in Example 1.

Step 8: Targeted Enrichment

The purified product was used as a template, and the primers designed for the downstream exons of the target intron were used as a combination of targeted gene-specific primers and a universal sequencing primer (PE1_p26) for PCR reaction.

The components of the PCR reaction system are shown in Table 19, the nucleic acid sequence of the PE1_p26 primer is shown in SEQ ID NO: 6, and the qPCR reaction conditions are shown in Table 20. The primer sequences are shown in Table 21.

Table 19: PCR reaction system (25 μL/reaction)

Table 20: PCR reaction conditions

Table 21: Nucleic acid sequences of primers

Among them, steps 9-10 are performed the same as steps 8-9 in Example 1.

The high-throughput sequencing results showed that the correlation between the actual detection values of splicing isoform IVT products with different mixing ratios and the theoretical values of mixed samples was r>0.99. The specific experimental results are shown in Table 22 and Figure 5.

Table 22: Detection results of RNA simulations with different ratios of splice isoforms

Example 4: Detection of splicing isoforms in HeLa cell lines

The high-throughput sequencing platform described in Example 1 was used to detect splicing isoforms in the HeLa cell line.

Wherein, steps 1-2 are the same as those in Example 1.

In the targeted enrichment reverse transcription process of step 3, gene-specific primers are used as reverse transcription primers, and common dNTPs and 3'-modified dNTPs at a molar concentration ratio of 1/15 are added to the reverse transcription system for reverse transcription reaction.

The gene-specific primer sequences are shown in Table 23, the preparation of the gene-specific primer combination mixture is shown in Table 24, the components of the reverse transcription reaction system are shown in Table 25, and the reverse transcription reaction conditions are shown in Table 26.

Table 23: Reverse transcription reaction gene specific primer sequences

Table 24: Gene-specific primer mix preparation

Table 25: Transcription reaction system (20 μL/reaction)

Table 26: Reverse transcription reaction conditions

Wherein, steps 4-6 are the same as those in Example 1.

In the targeted enrichment process of step 7, ATP13A1 gene, CXXC1 gene, ECHDC2 gene, FGFRL1 gene, HMGN3 gene, KLHL17 gene, OSGEP gene, NAXD gene, LZTR1 gene, SELENBP1 gene The primers designed for the downstream exons of the JMJD8 and JMJD9 genes were used as the targeted enrichment primer combination and the universal sequencing primer PE1_p26 for PCR reaction. Compared with the gene-specific primers in reverse transcription, the primers for targeted enrichment PCR are closer to the intron by about 20-50bp. The primer sequences of each targeted gene are shown in Table 27, the preparation of the primer mixture is shown in Table 28, the components of the PCR reaction system are shown in Table 29, the nucleic acid sequence of the PE1_p26 primer is shown in SEQ ID NO: 6, and the PCR reaction conditions are shown in Table 30.

Table 27: Primer combination mixture preparation

Table 28: Primer mix preparation

Table 29: PCR reaction system (25 μL/reaction)

Table 30: PCR reaction conditions

Steps 8-10 are the same as those in Example 1.

The final product was subjected to high-throughput sequencing, and the results of high-throughput sequencing showed that normal spliceosomes and splice isoforms (intron retention) were visible in the target genes of the HeLa cell line, and the ratio of splice isoforms in the targeted genes was successfully quantitatively detected. The specific test results are shown in Table 31. The above data show that the targeted high-throughput sequencing platform constructed in the present disclosure can effectively realize the detection of splice isoforms.

Table 31: Detection results of target genes in HeLa cell line

Example 5: Evaluation of trans-splicing off-target events

The high-throughput sequencing platform described in Example 1 was used to detect on-target trans-splicing and off-target trans-splicing events that occurred after HEK293T cells were transfected with mini-genes and trans-splicing factors (Pre-mRNA Trans-splicing Molecule).

Wherein, steps 1-2 are the same as those in Example 1.

In the targeted enrichment reverse transcription process of step 3, the reverse splicing molecule and the mini-gene specific primer are used as reverse transcription primers, and ordinary dNTP and 3' modified dNTP at a molar concentration ratio of 1/15 are added to the reverse transcription system for reverse transcription reaction.

The gene-specific primer sequences are shown in Table 32, the preparation of the gene-specific primer combination mixture is shown in Table 33, the components of the reverse transcription reaction system are shown in Table 34, and the reverse transcription reaction conditions are shown in Table 35.

Table 32: Reverse transcription reaction gene specific primer sequences

Table 33: Gene-specific primer mix preparation

Table 34: Transcription reaction system (20 μL/reaction)

Table 35: Reverse transcription reaction conditions

Wherein, steps 4-6 are the same as those in Example 1.

In the targeted enrichment process of step 7, the trans-splicing molecule and mini-gene specific primers are used as the targeted enrichment primer combination and the universal sequencing primer PE1_p26 for PCR reaction. Compared with the specific primers in reverse transcription, the primers of trans-splicing molecules and mini-gene targeted enrichment PCR are closer to the 3'splicing site (3'splicing site) and 5'splicing site (5'splicing site) by about 20-50bp. The primer sequences of each targeted gene are shown in Table 36, the preparation of the primer mixture is shown in Table 37, the components of the PCR reaction system are shown in Table 38, the nucleic acid sequence of the PE1_p26 primer is shown in SEQ ID NO:6, and the PCR reaction conditions are shown in Table 39.

Table 36: Primer combination mixture preparation

Table 37: Primer mix preparation

Table 38: PCR reaction system (25 μL/reaction)

Table 39: PCR reaction conditions

Steps 8-10 are the same as those in Example 1.

The final product was subjected to high-throughput sequencing, and the results of high-throughput sequencing showed that after HEK293T cells were transfected with mini-genes (mini-Gene) and trans-splicing factors (Pre-mRNA Trans-splicing Molecule), target trans-splicing products and off-target trans-splicing products were visible. At the same time, the genomic location where off-target trans-splicing of trans-splicing factors occurred was accurately determined (there were many events, not all of which were listed), and the specific test results are shown in Table 40. The above data show that the targeted high-throughput sequencing platform constructed in the present disclosure can effectively realize the evaluation of trans-splicing off-target events, and simultaneously perform qualitative and quantitative analysis of target trans-splicing and off-target trans-splicing.

Table 40: Detection results of trans-splicing and off-target trans-splicing in HEK293T cells

The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in the field, several modifications and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be subject to the attached claims.

Claims (18)

1. A method for establishing a sequencing library for high-throughput sequencing comprises the following steps:

   (1) reverse transcription of sample RNA using a reverse transcription primer, adding common dNTP and 3'-modified dNTP to obtain a first cDNA chain, wherein the 3'-modified dNTP is selected from one or more of the following: AzNTP, AmNTP, propargyl-NTP, HalNTP;

   (2) connecting the oligonucleotide fragment with an alkyne modification at the 5′ end to the cDNA fragment obtained in step (1) by a click chemistry reaction, wherein the oligonucleotide fragment comprises a random sequence and a complementary segment sequence of universal sequencing primer 1 (seq1);

   (3) using the reaction product of step (2) as a template, performing PCR amplification;

   (4) Obtain a sequencing library.
2. The method according to claim 1, wherein the reverse transcription primers in step (1) are random primers or gene-specific primer group 1 designed based on the downstream exons of the retained introns of the targeted gene;

   Optionally, the 5' end of the primer in the gene-specific primer group 1 carries a universal sequencing primer 2 sequence (seq2).
3. According to the method of claim 1 or 2, the PCR amplification primers used in step (3) include universal sequencing primer 1 and gene-specific primer group 2, and the 5' end of the primer in the gene-specific primer group 2 carries the universal sequencing primer 2 sequence.
4. According to the method of claim 2 or 3, both gene-specific primer groups 1 and 2 are designed based on the exons downstream of the selective splicing event of the retained intron of the specific targeted gene, wherein the targeting site of gene-specific primer 2 is shifted upstream by 5-100 bases, preferably 20-50 bases, than the site of gene-specific primer 1.
5. According to the method according to any one of claims 2 to 4, the number of target genes targeted by gene-specific primer group 1 and gene-specific primer group 2 respectively is greater than or equal to 1.
6. The method according to any one of claims 1 to 5, wherein the molar concentration ratio of the common dNTP and the 3'-modified dNTP added in step (1) is 1:1-1:100.
7. The method according to any one of claims 1 to 6, wherein the molar concentration ratio of the common dNTP and the 3'-modified dNTP added in step (1) is 1:20.
8. The method according to any one of claims 1 to 7, wherein the random sequence in step (2) comprises 4 to 16 nucleotides.
9. The method according to any one of claim 8, wherein the random sequence in step (2) is 5 nucleotides in length.
10. The method according to any one of claims 1 to 9, wherein the PCR amplification in step (3) comprises the step of performing PCR amplification on the reaction product of step (2) using a universal sequencing primer 1 and a gene-specific primer group 2, and then adding a linker structure to the amplified product, wherein the linker structure comprises a P5/P7 linker and a nucleic acid barcode, and the nucleic acid barcode is connected to the primer P5 and/or P7 linker end.
11. According to the method of claim 1, step (3) is: using the reaction product of step (2) as a template, adding P5 and P7 linkers to carry out PCR reaction.
12. The method according to any one of claims 1-11, wherein the universal sequencing primer 1 sequence is selected from any one of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 6; preferably, the complementary segment sequence of the universal sequencing primer 1 is as shown in SEQ ID NO: 4.
13. The method according to any one of claims 1-11, wherein the universal sequencing primer 2 sequence (seq2) is selected from any one of SEQ ID NO: 7 or SEQ ID NO: 8.
14. A high-throughput sequencing library constructed according to the method according to any one of claims 1 to 13.
15. A targeted high-throughput sequencing method for detecting splicing isoforms, comprising the following steps:

    (1) extracting sample RNA and constructing a sequencing library according to any one of claims 1 to 13;

    (2) Based on the above sequencing library, high-throughput sequencing is performed to obtain sequencing information of the target gene in the sample.
16. The method according to claim 15, wherein the sample is selected from the following cell samples: MCF10A, MCF7, HeLa, HEK293T and/or MDA-MB-231.
17. The method according to claim 15 or 16, wherein the target gene includes ATP13A1, CXXC1, ECHDC2, FGFRL1, HMGN3, KLHL17, NAXD, LZTR1, SELENBP1, JMJD8, PSMB1, HIGD2A, HNRNPAB, SMARCC1, ATP5IF1, HIGD2B, RPS21, UQCC5, NFATC3, PCNP and/or OSGEP.
18. Use of the method for establishing a sequencing library for high-throughput sequencing according to any one of claims 1 to 13, the high-throughput sequencing library according to claim 14, and/or the targeted high-throughput sequencing method according to claims 15 to 17 in the assessment of off-target events;

    Preferably, the off-target event assessment includes the following:

    (1) Accurately determine the genomic location where off-target trans-splicing occurs in trans-splicing factors;

    (2) Quantitative analysis of on-target trans-splicing and off-target trans-splicing.