WO2025000136A1 - Method for preparing strand-specific library for rapid detection of various types of rnas, and high-throughput sequencing technique - Google Patents

Abstract

Provided in the present invention are a method for preparing a strand-specific library for the rapid detection of various types of RNAs, and a high-throughput sequencing method. A polyadenylic acid (poly A) tail is artificially added at the 3' terminal of various RNAs by using a poly A polymerase, and a single-stranded cDNA is synthesized using a poly-deoxythymidine ribonucleotide primer with deoxyuridine under the action of a reverse transcriptase. The obtained single-stranded cDNA molecule is subjected to a series of reactions, and the reaction product is finally amplified by means of PCR to obtain a strand-specific library of various types of RNAs. The library sample can be subjected to on-machine sequencing.

Description

A strand-specific library preparation method and high-throughput sequencing technology for rapid detection of multiple types of RNA Technical Field

The present invention relates to the field of biotechnology, and in particular to a method for preparing a chain-specific library for rapidly detecting multiple types of RNA and a high-throughput sequencing technology.

Background Art

With the development of basic disciplines such as molecular biology and genomics, liquid biopsy has become a hot spot in the field of precision medicine. Cell free RNA (cfRNA) exists in various body fluids, such as blood, urine, cerebrospinal fluid, saliva, milk, pleural effusion, ascites, etc., and can indicate and monitor the physiological state of the body. The types of RNA in cells include ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), micro non-coding RNA (miRNA), long non-coding RNA (lncRNA), etc. The free RNA in body fluid samples carries real-time gene expression information from different tissues and organs, which can provide more solutions for early screening of diseases, auxiliary diagnosis, recurrence monitoring, disease prognosis, etc. Due to the non-invasive or minimally invasive sampling method and the advantages of real-time and comprehensive monitoring, free RNA may become an important detection method for precision medicine in the future.

In order to fully explore the biomarker potential of free RNA, efficient, rapid, low-cost and stable free RNA detection technology is needed. RNA detection technology based on high-throughput sequencing has the advantages of single-base resolution, wide detection range and high throughput. However, there are still many challenges in the preparation technology of free RNA high-throughput sequencing libraries, including but not limited to low sample starting amount, easy RNA degradation, long experimental cycle, low efficiency, single or limited target RNA type and other problems. At present, there are no commercial sequencing library preparation kits specifically for free RNA on the market. The scientific research field mainly uses kits suitable for sequencing library preparation of conventional cell tissue sample RNA to study free RNA. The commercial RNA sequencing library preparation kits on the market have relatively single or limited target RNA types, and the information that can be obtained is also limited, and it is impossible to conduct systematic research on all types of RNA.

Currently, there are no commercial library preparation kits on the market that can comprehensively capture multiple types of RNA and specifically target free RNA. The alternative solution is to use conventional RNA sequencing library preparation kits to construct sequencing libraries for free RNA.

Conventional RNA sequencing library preparation kits are mainly divided into kits suitable for mRNA and miRNA, which cannot capture multiple types of RNA in total RNA at the same time. Various types of RNA include mRNA, lncRNA, tRNA, miRNA, etc. Most commercial RNA sequencing library preparation kits have high requirements for the starting amount of total RNA. In addition, commercial RNA sequencing libraries have more experimental steps, more complicated processes, and longer experimental cycles, which are not conducive to rapid and stable detection in the clinical field. Currently, commercial sequencing library preparation kits suitable for mRNA detection include Illumina's TruSeq RNA Library Prep Kit v2, TruSeq Stranded mRNA and Total RNA Library prep kits, NEB's NEBNext Ultra ^TM II RNA Library Prep Kit for Illumina, TAKARA's SMARTer Stranded Total RNA-Seq Kit, etc. The above kit scheme uses polydeoxythymidine ribonucleotide (oligo dT) magnetic beads or primers to selectively enrich mRNA containing polyadenylic acid (polyA) tails and specifically prepare mRNA sequencing libraries. The above kit can also capture lncRNA while capturing mRNA by adapting the rRNA removal step and then using primers containing multiple random bases for reverse transcription, but this method is difficult to capture more short fragments of miRNA and other RNA. The above kits all need to first reverse transcribe into single-stranded cDNA, and then synthesize complementary double-stranded DNA to form double-stranded DNA, and then prepare it as a sequencing library on this basis, so the experimental steps are relatively complicated and the experimental cycle is relatively long.

There are also many commercial kits suitable for miRNA or small RNA. For example, Illumina's TruSeq small RNA Library Prep Kit, Bioo Scientific's Nextflex small RNA-Seq Kit v3, NEB's NEBNext small RNA Library Prep Set for Illumina, MGI's MGIEasy small RNA library preparation kit, and QIAGEN's QIAseq miRNA Library Kit. The above kits all use RNA ligase to directly connect adapters containing specific sequences to the 3' and 5' ends of RNA molecules, then transcribe them into cDNA, and then prepare sequencing libraries through PCR amplification reactions. The above kits are not suitable for capturing long-chain RNAs such as mRNA and lncRNA, and the experiments require RNA ligase, which is relatively costly, relatively inefficient, takes a long time to connect, has many steps, and a long experimental cycle.

There is a commercial kit for small RNA, SMARTer smRNA-Seq Kit for Illumina, which has a special principle. First, polyadenylic acid tailing is performed on the 3’ end of the small RNA, and then reverse transcription is performed using primers containing adapter sequences and polydeoxythymidine ribonucleotides (oligo dT), and the template switching method of reverse transcription is used to introduce the adapter sequence at the other end at the end of the cDNA. Finally, PCR is performed using the adapter sequences added at both ends to complete the preparation of the sequencing library. However, the above method will introduce artificially added polydeoxythymidine ribonucleotide sequences, which reduces the complexity of the library sequence and will seriously affect the sequencing quality. In order to reduce the impact of low-complexity sequences on sequencing, it is necessary to add a base-balanced library during sequencing, but this solution will result in a reduction in the amount of effective data.

Commonly used RNA library preparation methods are not suitable for low starting amounts or degraded samples, and have a low adaptability to plasma samples. Scientific research programs generally require mL-level plasma inputs, and this higher starting amount of plasma input will reduce its widespread use.

Summary of the invention

In view of this, the technical problem to be solved by the present invention is to provide a method for preparing a chain-specific library for rapidly detecting multiple types of RNA and a high-throughput sequencing technology. The present invention provides a method for preparing a chain-specific library for rapidly detecting multiple types of RNA and a high-throughput sequencing technology, which have high sensitivity, a wide range of applications, strong anti-interference ability, a simple and quick preparation method, and are suitable for high-throughput sequencing technology.

The present invention provides a method for preparing various types of RNA libraries, including: adding polyA to the end of an RNA sample, performing reverse transcription and U base digestion to prepare a cDNA library;

The RNA sample contains at least one of total RNA, rRNA, mRNA, tRNA, miRNA and/or lncRNA.

The present invention adds polyadenylic acid (poly A) tails to the ends of various types of RNA molecules and combines the reverse transcription method using polydeoxythymidine ribonucleotide (oligo dT) primers, so that various types of RNA molecules including non-coding RNA (lncRNA, miRNA, etc.) can be reverse transcribed in subsequent steps, thereby realizing the construction of libraries of various types of RNA.

Furthermore, the preparation method specifically includes: DNA digestion, RNA end modification, poly A tail addition, reverse transcription, U base digestion, cDNA end modification, denaturation, linker addition and library construction.

The present invention combines two or more of the above-mentioned specific reaction steps by optimizing the reaction reagents and other conditions. For example, in some embodiments, the steps of DNA digestion, RNA end modification and polyA tailing are combined. In some embodiments, the steps of DNA digestion and RNA end modification are combined, and then the steps of polyA tailing and reverse transcription are combined. In some embodiments, the steps of U base digestion, cDNA end modification and denaturation are combined. In some embodiments, the steps of U base digestion and cDNA end modification are combined. By combining the reaction steps, the reaction steps are reduced and the required time is shortened, while ensuring the efficiency of the reaction and the quality of the obtained library.

In some embodiments, the DNA digestion and RNA end modification are performed in a first system;

The reverse transcription is performed in a second system;

The step of adding the polyA tail is performed in the first system or the second system.

Furthermore, in a specific embodiment,

The step of adding polyA tail is carried out in a first system, wherein:

The first system includes: RNA sample, PolyA polymerase reaction buffer, ATP, BSA, DNase I, T4 polynucleotide kinase, PolyA polymerase, RNase inhibitor and nuclease-free water;

The second system includes: the reaction product of the first system, reverse transcription primer, HiScript III reaction buffer, HiScript III reverse transcriptase, dNTP Mix, RNase inhibitor and nuclease-free water.

In order to ensure that RNA terminal modification, addition of ployA tail structure and reverse transcription can react smoothly in the same system, the present invention optimizes the reaction system. The components in the reaction system are properly coordinated to jointly ensure the progress of the reaction. Furthermore, the present invention optimizes the concentration of the components in each system.

The concentrations of the components in the first system are as follows: 14 μL RNA sample, 3 μL 10×PolyA polymerase reaction buffer, 1 μL 10 mM ATP, 4 μL 10 mg/mL BSA, 2 μL DNase I, 0.5 μL 10 U/μL T4 polynucleotide kinase, 1 μL 5 U/μL PolyA polymerase, 0.5 μL 40 U/μL RNase inhibitor, and 4 μL nuclease-free water;

The concentrations of the components of the second system include: 30 μL of the reaction product of the first system, 2 μL of 5 μM reverse transcription primer, 4 μL of 5×HiScript III reaction buffer, 1 μL of 200 U/μl HiScript III reverse transcriptase, 2 μL of 5 mM dNTP Mix, 0.5 μL of 40 U/μl RNase inhibitor and 10.5 μL of nuclease-free water.

Furthermore, in another specific embodiment,

The step of adding polyA tail is carried out in the second system, wherein:

The first system includes: RNA sample, DNase I reaction buffer, ATP, DNase I, T4 polynucleotide kinase and RNase inhibitor;

The second system includes: the reaction product of the first system, HiScript III reaction buffer, BSA, PEG8000, dNTP Mix, reverse transcription primer, PolyA polymerase, RNase inhibitor, HiScript III reverse transcriptase and nuclease-free water.

In order to ensure that DNA digestion and RNA end modification react smoothly in one system, and that adding a ployA tail structure and reverse transcription react smoothly in the same system, the present invention optimizes the reaction system. The components in the reaction system are properly coordinated to jointly ensure the progress of the reaction. Furthermore, the present invention optimizes the concentration of the components in each system.

The concentrations of the components in the first system are: 14 μL RNA sample, 2 μL 10× DNase I reaction buffer, 1 μL 10 mM ATP, 2 μL DNase I, 0.5 μL 10 U/μL T4 polynucleotide kinase, and 0.5 μL 40 U/μL RNase inhibitor.

The concentrations of the components of the second system are: 20 μL of the reaction product of the first system, 6 μL of 5×HiScript III reaction buffer, 1 μL of 10 mg/mL BSA, 10 μL of 50% PEG8000, 2 μL of 5 mM dNTP Mix, 2 μL of 5 μM reverse transcription primer, 1 μL of 5 U/μL Poly A polymerase, 0.5 μL of 40 U/μl RNase inhibitor, 1 μL of 200 U/μl HiScript III reverse transcriptase and 6.5 μL of nuclease-free water.

In some embodiments, the cDNA end modification and denaturation are performed in a third system.

The third system comprises: the reaction product after U base digestion, polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer with pH 8.0, ultra-thermostable single-stranded binding protein and nuclease-free water.

In order to smoothly react the cDNA end modification and denaturation in the same system, the present invention optimizes the reaction system. The components in the reaction system are properly coordinated to jointly ensure the progress of the reaction. Furthermore, the present invention optimizes the concentration of the components in each system.

The concentrations of the components of the third system are: the reaction product after U base digestion, 5 μL of 10× polynucleotide kinase reaction buffer, 1 μL of 10U/μL T4 polynucleotide kinase, 2 μL of 220mM Tris buffer, pH 8.0, 0.6 μL of 500ng/μL ultra-thermostable single-stranded binding protein, and 21.4 μL of nuclease-free water.

In other embodiments, cDNA end modification, denaturation and U base digestion are performed in the same system, and therefore, the third system also includes reagents for U base digestion.

In this embodiment, the third system includes: the reaction product of polyA tailing and reverse transcription, polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer at pH 8.0, ultra-thermostable single-stranded binding protein, uracil-specific excision reagent USER enzyme and nuclease-free water.

In order to smoothly react the cDNA end modification and denaturation in the same system, the present invention optimizes the reaction system. The components in the reaction system are properly coordinated to jointly ensure the progress of the reaction. Furthermore, the present invention optimizes the concentration of the components in each system.

The concentrations of the components of the third system are: the reaction product after polyA tailing and reverse transcription, 5 μL of 10× polynucleotide kinase reaction buffer, 1 μL of 10 U/μL T4 polynucleotide kinase, 2 μL of 220 mM Tris buffer, pH 8.0, 0.6 μL of 500 ng/μL ultra-thermostable single-stranded binding protein, 2 μL of 10 U/μL uracil-specific excision reagent USER enzyme, and 19.4 μL of nuclease-free water.

Compared with the prior art, the present invention adopts a flexible method of combining multiple reaction steps into the same experimental operation step and optimizes the experimental conditions, thereby reducing the operation steps and time required for library preparation and ensuring the efficiency of the reaction, thereby obtaining better experimental results.

Further, the reverse transcription primer has the following nucleotide sequence: poly(T)n-UVNm;

Where n represents the number of bases T, and m represents the number of bases N;

n is an integer of 8 to 50, and m is an integer of 1 to 4

At least one T in the poly(T)n is replaced by U; V is selected from any one of base A, base C and base G; and N is selected from any one of base A, base T, base C and base G.

The present invention provides a polydeoxythymidine ribonucleotide (oligo dT) reverse transcription primer containing deoxyuracil (dU) for synthesizing a single-stranded cDNA molecule by reverse transcription reaction. The primer sequence comprises deoxyuracil ribonucleotide (dU), polydeoxythymidine ribonucleotide (oligo dT), and other random bases (V and N). By using the reverse transcription primer provided by the present invention, a single-stranded cDNA molecule containing a polydeoxythymidine ribonucleotide sequence of deoxyuracil (dU) can be obtained, so that a uracil-specific excision reagent is used to perform a digestion reaction on the single-stranded cDNA in the U base digestion step, thereby excising the polydeoxythymidine ribonucleotide sequence fragment containing deoxyuracil (dU) in the single-stranded cDNA, removing the influence of the low-complexity sequence artificially introduced in the previous step on the subsequent sequencing and analysis, and better realizing the preparation of the cDNA library. Compared with the prior art that artificially introduces low-complexity sequences to capture miRNA or single-stranded DNA and adds base-balanced libraries during sequencing (such as the SMARTer smRNA-Seq Kit for Illumina), the solution provided by the present invention is more conducive to increasing the amount of effective data in sequencing.

In some specific embodiments, the reverse transcription primer has a nucleotide sequence as shown in SEQ ID NO: 1. Experiments have shown that, compared with other reverse transcription primers, for example, primers with N being 2, 3 or 4, the reverse transcription primer shown in SEQ ID NO: 1 can achieve a better reverse transcription reaction, thereby obtaining a better experimental effect.

Furthermore, in the step of adding a linker, the linker includes a 5' end linker and a 3' end linker, the 5' end linker sequence has a nucleotide sequence as shown in SEQ ID NO 2 and SEQ ID NO 3, and the 3' end linker sequence has a nucleotide sequence as shown in SEQ ID NO 4 and SEQ ID NO 5.

The present invention provides a connector for introducing a specific target sequence at the 3' and 5' ends of a single-stranded cDNA molecule and a corresponding DNA connection scheme. Both connectors contain a double-stranded region and at least one protruding single-stranded region, wherein the double-stranded region contains a universal structural sequence for PCR or sequencing, and the protruding single-stranded region contains one or more (1 to 10) random base sequences, which are used for complementary pairing with the end of the single-stranded DNA molecule, so that the connector and the single-stranded DNA can be splinted. Each connector is formed by the interaction of two polynucleotide sequences to form its special structure, and the two sequences contain complementary pairing regions, which will be complementary paired to form a specific double-stranded structure and a protruding single-stranded region containing random bases after solution mixing and static treatment. After adding connectors at both ends of the single-stranded cDNA molecule, PCR amplification can be directly performed to obtain the final library, so as to better realize the preparation of cDNA library. Compared with the prior art solution that requires connecting two adapter sequences at the 3’ and 5’ ends of the DNA molecule in steps (such as SPLAT (Splinted ligation adapter tagging)), the present invention can connect the DNA adapters at the 3’ and 5’ ends simultaneously in one step, thereby shortening the steps and time while ensuring the connection effect.

In the present invention, the reaction system for adding a linker includes: a 5' end linker solution, a 3' end linker solution, a ligation reaction buffer, a ligation reaction enhancer and T4 DNA ligase.

In the reaction system for adding a linker described in the present invention, a DNA ligase scheme is used to replace RNA ligase, thereby greatly shortening the connection time (in the scheme of adding linkers at both ends of RNA using RNA ligase, due to the relatively low connection efficiency of RNA ligase, the single-end linker connection reaction time usually takes 1 to 2 hours, and the experimental steps of connecting linkers at both ends in steps are also relatively complicated, and the total connection time of the two-end linkers is 2 to 3 hours). In order to enable the DNA ligase to work better, the present invention optimizes other components and their concentrations in the reaction system, thereby further improving the connection efficiency of the linker.

In some embodiments, the reaction system for adding a linker includes: a 5' end linker solution, a 3' end linker solution, a ligation reaction buffer, a hexammaminecobalt chloride solution and T4 DNA ligase.

The present invention also provides a sequencing method for various types of RNA libraries, and the cDNA library prepared by the above preparation method is used as a sample for on-machine sequencing.

Furthermore, the cDNA library is sequenced after PCR amplification, purification, sample mixing, and single-stranded circularization.

In some specific embodiments, the upstream primer of the PCR amplification has a nucleotide sequence as shown in SEQ ID NO 6, and the downstream primer of the PCR amplification has a nucleotide sequence as shown in SEQ ID NO 7.

The downstream primers may contain a label (Barcode) sequence for sample identification. By using the primer sequence to amplify the library sample, library samples with different Barcode sequence labels can be obtained. At the same time, the Barcode sequence of the primer band can be matched with the Barcode of the universal sequencing library of the sequencing platform. The structure of the PCR amplification product can be consistent with the universal library structure of the sequencing platform, so that the source of the sample library can be accurately separated by the Barcode.

The present invention provides library construction reagents, including reagent I, reagent II and reagent III;

The reagent I comprises: DNase I reaction buffer, ATP, DNase I, T4 polynucleotide kinase, and RNase inhibitor;

The reagent II includes: reverse transcription primer, HiScript III reaction buffer, HiScript III reverse transcriptase, dNTP Mix, and RNase inhibitor;

The reagent III comprises: polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer at pH 8.0, and super-thermostable single-stranded binding protein.

In some embodiments, the reagent I further comprises: PolyA polymerase reaction buffer, BSA, PolyA polymerase;

In some embodiments, the reagent II also includes: BSA, PEG8000, and PolyA polymerase.

In some embodiments, the reagent III also includes a U base excision reagent, and the U base excision reagent includes a uracil-specific excision reagent USER enzyme.

Furthermore, it also includes a linker adding reagent, which includes: Tris-Hcl buffer, sodium chloride, EDTA, linkers as shown in SEQ ID NO 2 to 5, ligation reaction buffer, T4 ligase and hexamminecobalt chloride.

Furthermore, it also includes a purification reagent, which includes Agencourt AMPure XP magnetic beads.

Furthermore, it also includes PCR amplification reagents, which include PCR enzyme reaction solution, upstream primers of the nucleotide sequence shown in SEQ ID NO 6, and downstream primers of the nucleotide sequence shown in SEQ ID NO 7.

Furthermore, it also includes RNA extraction reagents.

The present invention also provides the application of the construction reagent in preparing various types of RNA libraries.

The present invention provides a method for preparing a strand-specific library for rapid detection of multiple types of RNA and a high-throughput sequencing technology. The present invention uses polyadenylic acid polymerase to artificially add polyadenylic acid (poly A) tails to the 3' ends of multiple RNAs, and simultaneously uses polydeoxythymidine ribonucleotide primers with deoxyuracil to synthesize single-stranded cDNA under the action of reverse transcriptase. The obtained single-stranded cDNA molecules are subjected to a series of reactions and finally amplified by PCR to obtain a strand-specific library of multiple types of RNA. Compared with the prior art:

(1) The present invention uses polyadenylic acid polymerase to artificially add polyadenylic acid (poly A) tails to the 3’ ends of various RNAs, so that various types of RNA molecules including non-coding RNA (lncRNA, miRNA, etc.) can be reverse transcribed in subsequent steps, thereby achieving library construction and sequencing of various types of RNAs;

(2) Single-stranded cDNA is synthesized using poly-deoxythymidine ribonucleotide primers containing deoxyuracil under the action of reverse transcriptase. In the subsequent reaction, the obtained single-stranded cDNA molecules can remove the artificially added polynucleotide sequences through the U base digestion reaction, avoiding the addition of low-complexity nucleotide sequences in the sequencing library, so that the sequencing quality and data volume are guaranteed;

(3) The present invention adopts a single-stranded cDNA molecule library preparation method, which does not require the synthesis of complementary double-stranded DNA of cDNA molecules, thereby reducing the steps and time required for library preparation. At the same time, since the present invention adopts a single-stranded cDNA molecule library preparation method, the strand-specific information of RNA is retained, which is more conducive to RNA analysis such as gene annotation;

(4) The present invention combines some reaction steps by optimizing the reaction reagents and other conditions. For example, in Example 3, the three reaction steps of DNA digestion, RNA end modification and polyadenylic acid tailing reaction are combined into the same operation step; the two reaction steps of cDNA end modification and denaturation reaction are combined into one operation step. In Example 4, the two reaction steps of DNA digestion and RNA end modification reaction are combined into one operation step; the two reaction steps of polyadenylic acid tailing and reverse transcription reaction are combined into one operation step; the three reaction steps of U base digestion, cDNA end modification and denaturation reaction are combined into the same operation step, etc. The overall solution reduces the number of operation steps and time required for library preparation, and ensures the efficiency of the reaction;

(5) The scheme of adding connectors at both ends of the single-stranded DNA of the present invention adopts a one-step connection method, which can simultaneously connect the DNA connectors at the 3' and 5' ends, reducing the experimental steps and time. The present invention adds a connection reaction enhancer, such as the chemical reagent hexaaminocobalt chloride, to the connection reaction, which can significantly improve the reaction efficiency of the connection reaction and further shorten the connection reaction time;

(6) The present invention uses the reverse transcribed cDNA and the adapter for connection and adds the sequencing structure sequence. Since the DNA ligase scheme is used instead of the RNA ligase scheme, the DNA ligase is superior to the RNA ligase in terms of both cost and efficiency. The total connection time only takes 30 minutes or even shorter. The overall experimental process has fewer operating steps and takes less time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the RNA library preparation and detection principle;

FIG2 is a schematic diagram of linker preparation, wherein P represents a phosphorylation modification group and B represents a modification group that blocks the connection;

FIG3 is a schematic diagram showing a method for preparing an RNA library;

FIG4 shows the sequencing quality distribution diagram of RNA library sequencing along with the sequencing cycle number;

FIG5 shows the number of various RNA genes detected in the samples, wherein the RNA samples in FIG5a include mRNA IncRNA and pseudogene RNA, and the RNA samples in FIG5b include miRNA, tRNA, mt-tRNA, mt-rRNA, snoRNA and snRNA;

FIG6 shows the number and percentage of various RNA genes detected in the samples, wherein FIG6a is a 200 μL starting plasma free RNA sample, FIG6b is a 10 ng starting amount UHRR sample, and FIG6c is a 2 ng starting amount UHRR sample;

FIG7 shows a scatter plot of gene expression between samples and a consistency analysis result diagram, wherein the Log2(TPM+1) value of gene expression is used for analysis, FIG7a is two technical parallels of a 200 μL starting plasma free RNA sample, FIG7b is a UHRR sample with a starting amount of 10 ng and 100 ng, and FIG7c is a UHRR sample with a starting amount of 2 ng and 10 ng;

FIG8 shows the Pearson correlation coefficient results of gene expression between samples, wherein FIG8a is the Pearson correlation coefficient calculated using the TPM value of the gene expression, and FIG8b is the Pearson correlation coefficient calculated using the Log2(TPM+1) value after the gene expression is converted.

DETAILED DESCRIPTION

The present invention provides a method for preparing a strand-specific library and a high-throughput sequencing technology for rapid detection of multiple types of RNA. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

The present invention provides a chain-specific library preparation and high-throughput gene sequencing technology for rapid detection of various types of RNA molecules. This technology can read various types of RNA information with a relatively low starting amount of RNA input, and is suitable for various types of usage scenarios such as tissue cells and body fluid samples. Through special reverse transcription primer design and digestion treatment, the interference of artificially added low-complexity sequences on sequencing can be removed, and chain-specific RNA library preparation can be achieved using single-stranded library preparation technology. By introducing a barcode sequence for sample identification and a connector structure sequence for sequencing reaction, various types of RNA information can be read and analyzed using high-throughput sequencing technology.

The present invention provides a chain-specific library preparation method and high-throughput sequencing technology for rapid detection of multiple types of RNA, which can simultaneously detect multiple types of RNA including mRNA, lncRNA, tRNA, miRNA, etc. The method provided by the present invention is suitable for high-throughput sequencing detection of multiple sample types such as tissue cell RNA and free RNA (cell-free RNA, abbreviated as cfRNA), and can be applicable to RNA samples with low starting amounts at the ng level or even the pg level. The present invention can be used in the fields of free RNA molecular diagnosis in the field of liquid biopsy, RNA molecular detection in tissue cells, etc., and has broad application prospects in the directions of early disease screening, auxiliary diagnosis, recurrence monitoring, disease prognosis, etc., among which potential clinical application scenarios include but are not limited to complex diseases such as pregnancy diseases, tumors, cardiovascular and cerebrovascular diseases, infectious diseases, genetic diseases, nervous system diseases, and mental diseases. The present invention can be extended to research in the fields of animals, plants, and microorganisms, and simultaneously detect various types of RNA; it can be used to study the growth and development characteristics, tissue and cell functions, gene functions, environmental adaptability, species interactions, etc. of different species, and it also has application prospects in the fields of agriculture, food, and biosafety. The technical principle of the present invention may also be extended to single-cell or spatiotemporal omics technology, which can be used to simultaneously capture different types of RNA, including non-coding RNA, and characterize the expression of multiple types of RNA in single cells or at different spatial locations. The principle of the technology of the present invention, combined with long-chain PCR technology and single-molecule sequencing, may also be applied to the application research of the full-length transcriptome.

First, an optional step is to perform DNA digestion on the RNA sample to remove the influence of residual DNA molecules on subsequent steps. On this basis, the end modification enzyme is used to modify the end of the RNA molecule, and the 3' end of the RNA molecule is hydroxylated, so that more natural RNA molecules can achieve polyadenylic acid tailing reaction at the 3' end. Then, polyadenylic acid polymerase is used to complete polyadenylic acid tailing at the 3' end of the RNA molecule. Polydeoxythymidine ribonucleotide (oligo dT) primers containing deoxyuracil (dU) are used to complementally pair with RNA molecules, and single-stranded cDNA is synthesized using RNA as a template under the action of reverse transcriptase to obtain single-stranded cDNA molecules with deoxyuracil (dU) and polydeoxythymidine ribonucleotide (oligo dT) sequences. Next, a uracil-specific excision reagent is used to digest the cDNA, thereby excising the polydeoxythymidine ribonucleotide sequence fragment containing deoxyuracil (dU) in the cDNA, removing the influence of the low-complexity sequence artificially introduced in the previous step on subsequent sequencing and analysis. Then, the cDNA library is prepared.

The cDNA library preparation method provided by the present invention is to first perform high temperature denaturation treatment on cDNA to unwind the double-stranded structure that may appear in the local area of cDNA. The denaturation reaction system can add single-stranded binding protein to assist in maintaining the linear structure of single-stranded cDNA molecules to avoid annealing and renaturing into complex hairpin-like structures after denaturation. The cDNA after high temperature denaturation is then used through special double-stranded adapters and matching connection technology to complete the cDNA library preparation.

The present invention provides a single-stranded DNA library preparation technology. Through special double-stranded adapters and one-step connection technology, adapters can be added to both ends of the single-stranded cDNA molecule at the same time, and a universal structural sequence for PCR amplification reaction can be introduced, and then a sequencing library can be obtained through PCR amplification. The sequencing library product is subsequently processed and sequenced according to the requirements of the gene sequencer, and the purpose of detecting RNA is achieved by reading and analyzing the sequence information.

The schematic diagrams of the above library preparation are shown in Figures 1 and 3.

In the PCR amplification step of library preparation, the label (Barcode) sequence for sample identification can be introduced through PCR primers, and the library samples with different Barcode sequences can be mixed into a sequencing sample according to the proportion requirements of the sequencing data. The sequencing samples are subsequently processed and sequenced according to the requirements of the gene sequencer. Each sequencing result will be accurately located in each sample after the Barcode sequence alignment and splitting, and the sequencing results of each sample will be sequenced to achieve the purpose of detecting RNA for each sample. The Barcode sequence is used to achieve the mixing of multiple library samples for sequencing detection, improve the detection throughput, and reduce the detection cost.

The present invention completes polyadenylic acid tailing at the 3' end of various types of RNA molecules, and combines the reverse transcription method using polydeoxythymidine ribonucleotide (oligo dT) primers to achieve effective reverse transcription of various types of RNA, thereby achieving the capture of various types of RNA molecules and the construction of sequencing libraries. Various types of RNA include but are not limited to mRNA, lncRNA, miRNA, tRNA, etc. The polyadenylic acid tail is Poly (A) n (n represents the number of adenine A, and n is an integer between 8 and 200).

The present invention designs a polydeoxythymidine ribonucleotide (oligo dT) primer containing deoxyuracil (dU) for synthesizing a single-stranded cDNA molecule by reverse transcription reaction, and the primer sequence is named as the first nucleotide sequence. It should be noted that the primer sequence contains deoxyuracil ribonucleotide (dU), polydeoxythymidine ribonucleotide (oligo dT), and other random bases (V and N), and its sequence is the nucleotide composition shown in Table 1 below. By using the primer sequence of the present invention for reverse transcription reaction, a cDNA molecule containing a polydeoxythymidine ribonucleotide sequence of deoxyuracil (dU) can be obtained.

Table 1 Polydeoxythymidine ribonucleotide primer sequence structure with deoxyuracil (5'-3' direction)

By optimizing the experimental conditions, the present invention can combine the multiple reaction steps mentioned in the above experimental principles into the same experimental operation step, thereby reducing the operation steps and time required for library preparation and ensuring the efficiency of the reaction.

The preparation method of the library of the present invention specifically includes: DNA digestion, RNA end modification, adding polyA tail, reverse transcription, U base digestion, cDNA end modification, denaturation, adding linker and library construction.

In some embodiments of the present invention, the DNA digestion and RNA end modification are performed in a first system; the reverse transcription is performed in a second system; and the cDNA end modification and denaturation are performed in a third system.

Wherein, the step of adding the polyA tail is performed in the first system or the second system.

In some specific embodiments, the step of adding the polyA tail is performed in a first system, wherein the first system is: 14 μL of RNA sample, 3 μL of 10×PolyA polymerase reaction buffer, 1 μL of 10 mM ATP, 4 μL of 10 mg/mL BSA, 2 μL of DNase I, 0.5 μL of 10 U/μL T4 polynucleotide kinase, 1 μL of 5 U/μL PolyA polymerase, 0.5 μL of 40 U/μL RNase inhibitor, and 4 μL of nuclease-free water;

The concentrations of the components of the second system are: 30 μL of the reaction product of the first system, 2 μL of 5 μM reverse transcription primer, 4 μL of 5×HiScript III reaction buffer, 1 μL of 200 U/μl HiScript III reverse transcriptase, 2 μL of 5 mM dNTP Mix, 0.5 μL of 40 U/μl RNase inhibitor and 10.5 μL of nuclease-free water.

In some other specific embodiments, the step of adding polyA tail is performed in a second system, wherein the first system is: 14 μL RNA sample, 2 μL 10× DNase I reaction buffer, 1 μL 10 mM ATP, 2 μL DNase I, 0.5 μL 10 U/μL T4 polynucleotide kinase and 0.5 μL 40 U/μL RNase inhibitor;

The second system consists of: 20 μL of the reaction product of the first system, 6 μL of 5×HiScript III reaction buffer, 1 μL of 10 mg/mL BSA, 10 μL of 50% PEG8000, 2 μL of 5 mM dNTP Mix, 2 μL of 5 μM reverse transcription primer, 1 μL of 5 U/μL Poly A polymerase, 0.5 μL of 40 U/μl RNase inhibitor, 1 μL of 200 U/μl HiScript III reverse transcriptase and 6.5 μL of nuclease-free water.

In some specific embodiments, the third system is: the reaction product after U base digestion, 5 μL of 10× polynucleotide kinase reaction buffer, 1 μL of 10U/μL T4 polynucleotide kinase, 2 μL of 220mM Tris buffer, pH 8.0, 0.6 μL of 500ng/μL ultra-thermostable single-stranded binding protein, and 21.4 μL of nuclease-free water.

In some other specific embodiments, the third system also includes a reagent for U base digestion. The third system is: the reaction product of polyA tailing and reverse transcription, 5 μL of 10× polynucleotide kinase reaction buffer, 1 μL of 10U/μL T4 polynucleotide kinase, 2 μL of 220mM Tris buffer at pH 8.0, 0.6 μL of 500ng/μL ultra-thermostable single-stranded binding protein, 2 μL of 10U/μL uracil specific excision reagent USER enzyme, and 19.4 μL of nuclease-free water.

In the above library preparation method, while ensuring the library construction effect, different reaction steps can be selectively combined into the same experimental operation step, so as to flexibly modify the experimental steps and control the experimental time. The combination of reaction steps can be adjusted according to the specific experiment, and the present invention is not limited to this.

The present invention provides a connector for introducing a specific target sequence at the 5' and 3' ends of a single-stranded cDNA molecule and a corresponding DNA connection scheme. This scheme involves two connectors, a first connector and a second connector. Both connectors contain a double-stranded region and at least one protruding single-stranded region, wherein the double-stranded region contains a universal structural sequence for PCR or sequencing, and the protruding single-stranded region contains one or more (1-10) random base sequences, which are used for complementary pairing with the end of the single-stranded DNA molecule, so that the connector and the single-stranded DNA can be splinted. The protruding single-stranded region of the first connector is at the 5' end, and the protruding single-stranded region of the second connector is at the 3' end. The first connector is used for complementary pairing at the 5' end of the single-stranded DNA molecule, and the second connector is used for complementary pairing and splint connection at the 3' end of the single-stranded DNA molecule. One embodiment of the first connector is that two polynucleotide sequences interact to form its special structure, and the two sequences contain complementary pairing regions, which will be complementary paired to form a specific double-stranded structure and a protruding single-stranded region containing random bases after solution mixing and static treatment. One implementation of the second linker is that two polynucleotide sequences interact to form its special structure, and the two sequences contain complementary paired regions, which will complement each other to form a specific double-stranded structure and a protruding single-stranded region containing random bases after solution mixing and static treatment. The schematic diagram of the preparation principle of the first linker and the second linker solution is shown in Figure 2.

The present invention provides a sequence scheme of a first joint and a second joint, which consists of four nucleotide sequences, and the sequences are shown in Table 2. The first joint consists of two sequences, namely, the second nucleotide sequence and the third nucleotide sequence in Table 2. The 5' end of the third nucleotide sequence contains a random base sequence, the number of random bases is 1-10, and the random base is base A, base T, base C or base G; the random base sequence can be complementary to the 5' end of the single-stranded cDNA molecule in the first joint structure, and is used for splint connection to improve the connection efficiency. The two sequences of the second joint are the fourth nucleotide sequence and the fifth nucleotide sequence in Table 2. The 3' end of the fifth nucleotide sequence contains a random base sequence, the number of random bases is 1-10, and the random base is base A, base T, base C or base G; the random base sequence can be complementary to the 3' end of the single-stranded cDNA molecule in the second joint structure, and is used for splint connection to improve the connection efficiency. The 5' end of the second nucleotide sequence needs to be specially modified to block it from performing a connection reaction. The 5' end of the fourth nucleotide sequence is phosphorylated; the 3' end is specially modified to block the ligation reaction. The 5' end and 3' end of the third nucleotide sequence and the fifth nucleotide sequence are specially modified to block the ligation reaction. Under DNA ligase and appropriate reaction conditions, both the 5' and 3' ends of the single-stranded DNA molecule can be directly connected to the linker, and then the primers designed based on the linker sequence can be used for PCR amplification reaction.

The present invention uses a DNA ligation reaction enhancing reagent, for example, a chemical reagent hexaamminecobalt chloride with a suitable concentration is used to improve the DNA ligation efficiency, realize the connection of the two end adapters at the 5' end and the 3' end of the single-stranded cDNA molecule in a one-step experimental operation, and shorten the reaction time of the connection.

After adding adapters at both ends of the single-stranded cDNA molecule, PCR amplification can be directly performed to obtain the final library. Through PCR primers, a complete sequencing structure sequence can be introduced, including a barcode sequence that can distinguish samples, to adapt to the needs of the sequencing platform.

The present invention does not require an additional separate step to synthesize the complementary double-stranded DNA of the cDNA molecule, thereby reducing the steps and time required for library preparation. At the same time, since the present invention adopts the library preparation method of the single-stranded cDNA molecule, the strand-specific information of the RNA is retained, which is more conducive to RNA analysis such as gene annotation.

Table 2 Specially modified double-stranded DNA linker sequences containing random bases (5'-3' direction)

The present invention provides a set of universal primer sequences for PCR amplification of library samples, wherein the forward universal primer is the sixth nucleic acid sequence, and the reverse primer is the seventh nucleic acid sequence. The reverse primer may contain a label (Barcode) sequence for sample identification, which is named the seventh nucleic acid sequence-N, where N represents the Barcode number, and different Barcode sequences have different numbers. The universal primer sequence is composed of the nucleotides in Table 3 below. By using the primer sequence to amplify the library sample, a library sample with different Barcode sequence labels can be obtained.

By using the principles of the present invention, the Barcode sequence in the seventh nucleic acid sequence-N can be matched with the Barcode of the universal sequencing library of the sequencing platform, the structure of the PCR amplification product can be kept consistent with the universal library structure of the sequencing platform, and the source of the sample library can be accurately split by the Barcode.

Table 3 Universal primer sequences (5'-3' direction)

The adapter and PCR primer sequence schemes (Table 2 and Table 3) provided by the present invention are suitable for preparing high-throughput sequencing libraries of the DNBSEQ and MGISEQ series of the MGI sequencing platform of MGI. However, the design principle provided by the present invention can also be used to design sequence schemes suitable for other sequencing platforms to prepare compatible sequencing libraries.

The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

Example 1 Extraction and preparation of RNA samples

Universal Human Reference RNA (UHRR, Agilent, 740000) was extracted. One tube of commercial standard (200 μg RNA, 70% ethanol and 0.1 M sodium acetate solution) was centrifuged at 4°C, 12,000×g for 15 minutes, then the supernatant was aspirated and the precipitate was washed with 70% ethanol, centrifuged again at 4°C, 12,000×g for 15 minutes, the supernatant was carefully aspirated, the precipitate was dried at room temperature for 30 minutes, and the precipitate was re-dissolved with 1 mL of nuclease-free H ₂ O to a RNA concentration of about 200 ng/μL after re-dissolution, and the accurate concentration of the UHRR solution after re-dissolution was determined using a Qubit3.0 fluorescence quantifier (Invitrogen, Q33216). According to the measured concentration of UHRR solution, the standard solution was diluted stepwise with nuclease-free H ₂ O to prepare 4 samples with different RNA starting amounts, namely 100 ng, 10 ng, 2 ng and 0.2 ng, with a total volume of 14 μL in each tube.

A human plasma sample with a volume of 400 μL was used and divided into two portions of 200 μL plasma. Serum/plasma miRNA extraction and separation kits (spin column type, TIANGEN, DP503) were used for extraction, and the operation was performed strictly according to the instructions.

Example 2 Preparation of joint

2.1. Preparation of 5× STE buffer

According to the reaction system in Table 4, 5× STE buffer was prepared and thoroughly shaken to mix.

Table 4 5×STE buffer

2.2. Preparation of the linker

Use 5×STE buffer to prepare the connector. According to the systems in Table 5 and Table 6, prepare the first connector solution and the second connector solution with a concentration of 25 μM, respectively. The connector solution is fully shaken and mixed, and left to stand at room temperature for 30 minutes to allow the connector nucleotide sequences to fully complement and pair. After the reaction is completed, use pH 8.0 TE buffer (AMBION, AM9849) to dilute the 25 μM first connector solution and the 25 μM second connector solution to 1 μM first connector solution and 1 μM second connector solution, respectively, and store them at -18°C to -22°C. When using the connector, melt the connector solution at room temperature, shake it thoroughly, and then place it at 4°C for use. The schematic diagram of the preparation principle of the above-mentioned first connector and second connector solutions is shown in Figure 2.

Table 5 First linker (25 μM) solution configuration system

Table 6 Second linker (25 μM) solution configuration system

Example 3 RNA library preparation and high-throughput sequencing

The schematic diagram of library preparation is shown in Figure 1 and Figure 3. The specific experimental steps are described below.

3.1 RNA sample description

According to the method described in Example 1, the starting amounts of 2 ng, 10 ng, and 100 ng of human universal RNA standard UHRR solutions were prepared, and the samples were named UHRR-2ng, UHRR-10ng, and UHRR-100ng.

3.2 DNA digestion, RNA end modification and polyadenylation tailing reaction

DNase I (RNase Free) (NEB, M0303L) was used for DNA digestion to remove possible residual DNA in RNA preparation; T4 polynucleotide kinase (T4 PNK, NEB, M0201L) was used to modify the end of RNA molecules; polyadenylic acid tailing was performed at the 3' end of RNA molecules using polyadenylic acid polymerase (NEB, E. coli Poly (A) Polymerase, M0276L); the reaction system is shown in Table 7. The reaction system was incubated at 37°C for 15 minutes, inactivated at high temperature (95°C) for 5 minutes, and after the reaction, the sample was placed on ice for 2 minutes.

Table 7 DNA digestion, end modification and polyadenylation tailing reaction

3.3 Reverse transcription reaction

Add 2 μL of the first nucleic acid sequence primer (i.e., reverse transcription primer) at a concentration of 5 μM to the reaction product of the previous step. The first nucleotide sequence in this embodiment is specifically 5'-TTTTTTTUTTTTTTTUVN-3' (SEQ ID NO: 1). After adding the primer solution, shake and mix, centrifuge briefly, denature at 65°C for 5 minutes, and incubate at 30°C for 1 minute.

200 U/μl HiScript III Reverse transcriptase (Novozyme, R302-01) was used for reverse transcription reaction. 18 μl of reverse transcription reaction mixture was added to the reaction system, and its composition is shown in Table 8. The reaction system was placed in a PCR instrument (Bio-Tech, TC-96) and the program in Table 9 was run.

Table 8 Reverse transcription reaction mixture composition.

Table 9 Reverse transcription reaction procedure

3.4 Reverse transcription product purification

Add 100 μL of Agencourt AMPure XP magnetic beads (Beckman Coulter, A63881) to the reaction tube to purify the reverse transcription product, elute with 22 μL of pH 8.0 TE buffer (AMBION, AM9849), and take 20 μL of the purified ligation product to a new PCR reaction tube. Reserve it for library preparation.

3.5 U base digestion reaction

The U base digestion reaction was performed using uracil specific excision reagent (USER) enzyme (NEB, USER Enzyme, M5505L), and the digested single-stranded cDNA molecule product was obtained. 4 μL of U base digestion reaction mixture was added to the reaction system, and its composition is shown in Table 10. The reaction system was placed on a PCR instrument (Bori, TC-96) and the program in Table 11 was run.

Table 10 U base digestion reaction mixture composition.

Table 11 U base digestion reaction program

3.6 cDNA end modification and denaturation reaction

Add the end modification and denaturation reaction mixture to the reaction system, the composition of which is shown in Table 12. In the reaction system, T4 polynucleotide kinase (T4 PNK, NEB, M0201L) is used to phosphorylate the 5' end of the single-stranded cDNA molecule. Then, high temperature is used to denature the cDNA molecule to unwind the double-stranded structure that may appear in the local area of the cDNA. ET SSB (NEB, M2401S) is added to the denaturation reaction system to maintain the linear structure of the single-stranded cDNA molecule and avoid annealing and renaturation after denaturation to form complex hairpin-like structures. The reaction system is placed on a PCR instrument (Bori, TC-96) and the program in Table 13 is run.

Table 12. Composition of terminal modification and denaturation reaction mixture.

Table 13 Terminal modification and denaturation reaction procedures

3.7 Ligation reaction

2 μL of the first linker solution (1 μM) and 2 μL of the second linker solution (1 μM) prepared in Example 2 were added to the reaction system respectively, and 28 μL of the ligation reaction mixture was added, the composition of which is shown in Table 14. Among them, T4 DNA Ligase (NEB, M0202L) was used for linker connection, and the ligation reaction enhancer was 0.5 mM hexaamminecobalt chloride solution. The reaction system was placed on a PCR instrument (Bori, TC-96) and the program in Table 15 was run.

The present invention uses a DNA ligation reaction enhancing reagent. In this embodiment, a chemical reagent hexaamminecobalt chloride with a suitable concentration is used to improve the DNA ligation efficiency, realize the connection of the two end adapters of the 5' end and the 3' end of the single-stranded cDNA molecule in a one-step experimental operation, and shorten the reaction time of the connection.

Table 14 Composition of ligation reaction mixture

Table 15 Ligation Reaction Procedure

3.8 Purification of ligation product

Add 160 μL of Agencourt AMPure XP magnetic beads (Beckman Coulter, A63881) to the reaction tube, purify the ligation product, elute with 23 μL of pH 8.0 TE buffer (Invitrogen, AM9849), and take 21 μL of the purified ligation product to a new PCR reaction tube. Reserve it for universal PCR amplification.

3.9 Universal PCR Amplification

The primer of the sixth nucleic acid sequence (40 μM) is used as the forward primer, and the primer of the seventh nucleic acid sequence-N (40 μM) is used as the reverse primer for universal PCR amplification reaction. Among them, the reverse primer contains a Barcode sequence for sample identification. Each sample uses a unique Barcode sequence, and the different Barcode sequences in the reverse primers of different samples are used to distinguish different samples in the offline data. The PCR enzyme reaction solution used in this embodiment is KAPA HiFi HotStart ReadyMix (2X) (Kapa Biosystems, KK2602), and the reaction composition is shown in Table 16. The reaction system is placed on a PCR instrument (Bori, TC-96) to run the program in Table 17.

Table 16 General PCR amplification reaction solution components

Table 17 General PCR amplification reaction program

3.10 Universal PCR amplification product purification and pooling

50 μL of PCR product obtained by universal PCR amplification was purified using 50 μL of Agencourt AMPure XP magnetic beads (Beckman Coulter, A63881). The concentration of 35 μL of purified DNA was measured using Qubit3.0 fluorescence quantification instrument (Invitrogen, Q33216). At the same time, the library samples were mixed into sequencing library samples according to the same final concentration and shaken for use.

3.11 Single-stranded cyclization and sequencing reaction

Single-chain cyclization was performed using the MGIEasy cyclization module V2.0 (MGI, 1000005260) of Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., and sequencing was performed using the MGISEQ-2000RS high-throughput sequencing reagent kit (FCL PE100) (MGI, 1000012554) of Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd. All operations were performed strictly in accordance with the instructions of the kit. The PE100+10 (Paired end 100+10) sequencing type was used to obtain reliable base sequence information. The offline data was split and screened according to the Barcode sequence to obtain the sequencing data of each sample.

Example 4 Rapid preparation of RNA library and high-throughput sequencing

4.1 RNA sample description

According to the method described in Example 1, the human universal RNA standard UHRR was prepared with starting amounts of 0.2 ng, 2 ng, and 10 ng, respectively, and the samples were named UHRR-F-0.2 ng, UHRR-F-2 ng, and UHRR-F-10 ng.

According to the method described in Example 1, plasma free RNA samples were extracted and the samples were named Plasma-F-200uL-1 and Plasma-F-200uL-2.

4.2 DNA digestion and RNA end modification reactions

DNase I (RNase Free) (NEB, M0303L) was used for DNA digestion to remove the DNA that may remain after RNA preparation; T4 polynucleotide kinase (T4 PNK, NEB, M0201L) was used to modify the 3' end of RNA molecules. The reaction system is shown in Table 18. The reaction system was incubated at 37°C for 15 minutes and inactivated at high temperature (95°C) for 5 minutes. After the reaction, the sample was placed on ice for 2 minutes.

Table 18 DNA digestion and RNA end modification reactions

4.3 Polyadenylation and reverse transcription

Polyadenylic acid tailing was performed on the 3' end of the RNA molecule using polyadenylic acid polymerase (NEB, E. coli Poly (A) Polymerase, M0276L); reverse transcription was performed using the primer of the first nucleic acid sequence and 200U/μl HiScript III Reverse transcriptase (Novozyme, R302-01). The first nucleic acid sequence in this embodiment is specifically 5'-TTTTTTTUTTTTTTTUVN-3'. 30μL of polyadenylic acid tailing and reverse transcription reaction mixture was added to the reaction system, and its composition is shown in Table 19. The reaction system was placed on a PCR instrument (Bori, TC-96) and the program in Table 20 was run.

Table 19 Compositions of the polyadenylation tailing and reverse transcription reaction mixture.

Table 20 Polyadenylation tailing and reverse transcription reaction procedures

4.4 Reverse transcription product purification

Add 100 μL of Agencourt AMPure XP magnetic beads (Beckman Coulter Coulter, A63881) to the reaction tube, purify the reverse transcription product, elute with 22 μL of pH 8.0 TE buffer (Invitrogen, AM9849), and take 20 μL of the purified ligation product into a new PCR reaction tube. Reserve it for library preparation.

4.5 U base digestion, cDNA end modification and denaturation reaction

The U base digestion reaction was performed using uracil specific excision reagent (USER) enzyme (NEB, USER Enzyme, M5505L), and the digested single-stranded cDNA molecule product was obtained. The 5' end of the digested single-stranded cDNA molecule was phosphorylated using T4 polynucleotide kinase (T4 PNK, NEB, M0201L). The cDNA molecules were then denatured at high temperature to unwind the double-stranded structure that may appear in the local area of the cDNA. ET SSB (NEB, M2401S) was added to the denaturation reaction system to assist in maintaining the linear structure of the single-stranded cDNA molecules and avoid annealing and renaturing into complex hairpin-like structures after denaturation. 30 μL of the U base digestion reaction mixture was added to the reaction system, and its composition is shown in Table 21. The reaction system was placed on a PCR instrument (Bori, TC-96) and the program in Table 22 was run.

Table 21 U Base digestion, end modification and denaturation reaction mixture composition.

Table 22 U base digestion, end modification and denaturation reaction procedures

4.6 Ligation reaction

2 μL of the first linker solution (1 μM) and 2 μL of the second linker solution (1 μM) prepared in Example 2 were added to the reaction system respectively, and 28 μL of the ligation reaction mixture was added, the composition of which is shown in Table 14. Among them, T4 DNA Ligase (NEB, M0202L) was used for linker connection, and the ligation reaction enhancer was 0.5 mM hexaamminecobalt chloride solution. The reaction system was placed on a PCR instrument (Bori, TC-96) to run the program in Table 15.

4.7 Purification of ligation product

Add 80 μL of Agencourt AMPure XP magnetic beads (Beckman Coulter Coulter, A63881) to the reaction tube to purify the ligation product, elute with 23 μL of TE buffer (Invitrogen, AM9849), and take 21 μL of the purified ligation product to a new PCR reaction tube. Reserve it for universal PCR amplification.

4.8 According to steps 3.9, 3.10 and 3.11, complete universal PCR amplification, universal PCR amplification product purification and mixing, single-stranded circularization and sequencing reactions respectively.

Example 5 RNA sequencing data analysis and result presentation

The sequencing quality of the original data sequenced in Example 3 and Example 4 was analyzed, and the sequencing quality of the sequences above Q30 reached more than 90%. The quality of the sequencing chip was analyzed, and the sequencing quality did not decrease significantly during the entire sequencing cycle, and always remained at a high level, as shown in Figure 4. The above results show that high-quality sequencing data can be obtained using the method of the present invention.

The original sequencing data is split and screened by the Barcode sequence to obtain the sequencing data of each sample. The present invention can realize the simultaneous detection of multiple samples in the same sequencing chip.

The raw sequencing data (raw reads) of each sample were filtered for low-quality bases, short sequences, linkers, and microbial sequences, and then ribosomal RNA (rRNA) was removed. Using the human rRNA sequence as a reference, the rRNA sequence in the raw data was removed to obtain the filtered sequence (clean reads).

All filtered sequences (clean reads) were compared with the human genome (GRCh38) as a reference for RNA alignment. The number of clean reads, total alignment rate, unique alignment rate, and the ratio of alignment to exon regions were counted, as well as the positive and negative strand information of successfully aligned clean reads in the reference gene, so as to calculate the strand specificity ratio of the library. The results are shown in Table 23. The total alignment rate of most UHRR samples can reach 93%-97%, and the unique alignment rate can reach 60%-73%. The alignment rate of UHRR samples with low starting amount is relatively low, for example, the total alignment rate of UHRR-F-0.2ng is 43.63%, and the unique alignment rate is 35.27%; the total alignment rate of UHRR-F-2ng is 86.36%, and the unique alignment rate is 61.15%. The total alignment rate of plasma free RNA sample starting from 200μL is about 35%, and the unique alignment rate is about 28%. The chain-specific ratios calculated for UHRR samples were all above 80%, and most of them could reach around 90% or above; the chain-specific ratios calculated for plasma samples were around 70%.

The total number of genes detected in the sample and the number of genes of different types of RNA were counted. The RNA types analyzed in this embodiment include mRNA (messenger RNA), lncRNA (long non-coding RNA), pseudogene RNA, miRNA (microRNA), tRNA (transfer RNA), mt-tRNA (mitochondrial transfer RNA), mt-rRNA (mitochondrial ribosomal RNA), snoRNA (small neclear RNA), and snRNA (small cytoplasmic RNA). The results are shown in Figure 5. All UHRR samples, regardless of the starting amount of 0.2ng, 2ng, 10ng, or 100ng, can detect more than 10,000 protein-coding genes; can also detect about 4,000 lncRNA genes; can detect about 3,000 to 10,000 pseudogene RNA; can detect about 200-900 miRNA genes; can detect about 200-300 tRNA genes; can detect about 300-800 snoRNA genes and about 40-90 snRNA genes; 22 tRNAs and 2 rRNAs of mitochondria, namely mt-tRNA and mt-rRNA, can all be detected. For plasma samples starting with 200μL, the number of genes that can be detected in free RNA is relatively small compared to UHRR. But it can also detect nearly 10,000 protein-coding genes, and can detect other types of RNA. Attached Figure 6 of this embodiment also shows the percentage of the number of various RNA genes detected in some samples.

The quantitative analysis of RNA expression was performed using the TPM (Transcript per million) calculation method, based on the whole genome gtf (gene transfer format) file, to achieve qualitative and quantitative analysis of different types of RNA and obtain RNA expression profiles. The consistency of sample detection was analyzed using the RNA expression profile. As shown in the expression levels in Figure 7, both UHRR samples with different starting amounts and technical replicate samples of plasma free RNA have good consistency. For the expression levels between all samples, the Pearson correlation coefficient was calculated according to the original TPM value and the converted Log2 (TPM+1) value, and the results are shown in Figure 8. According to the Pearson correlation coefficient calculated from the original TPM value, the correlation coefficient between UHRR samples is at least 0.85; the correlation coefficient of the technical replicate samples of plasma free RNA is 0.99.

Quantitative analysis of RNA expression was performed according to the Log ² (TPM+1) value. The fluctuation of low-expression genes had a certain negative impact on the results, and the calculated correlation coefficient was lower than the value calculated by the original TPM value. The correlation coefficient of UHRR samples was at least 0.67, and most of them were above 0.70; the correlation coefficient of two technical replicates of plasma free RNA samples was 0.83.

It is worth mentioning that the UHRR sample starting amount range implemented in the present invention is relatively wide, including 4 different starting amount ranges of 0.2ng, 2ng, 10ng, and 100ng. Among them, the lowest UHRR starting amount is as low as 0.2ng. The starting amount of plasma samples is also as low as 200μL. The above starting amount is much lower than the RNA starting amount used in most of the current commercial RNA kits and literature. In the case of the low starting amount, under the experimental conditions of two batches of two different library preparation processes (Example 3 and Example 4), a high correlation coefficient can still be shown, indicating that the method of the present invention has high stability and low starting amount advantages.

Table 23 Basic information of sample sequencing data and alignment rate

In summary, the present invention is a strand-specific library preparation method and high-throughput sequencing technology for rapid detection of multiple types of RNA, and the RNA types that can be detected simultaneously include but are not limited to mRNA, lncRNA, tRNA, miRNA, etc. It solves the problems of the traditional RNA library preparation method that the RNA types captured are relatively single or limited, the experimental steps are complicated, and the time period is long.

The RNA sequencing library prepared by the present invention does not contain artificially added low-complexity sequences, so that the sequencing quality and data volume are guaranteed.

The sequencing library prepared by the present invention contains RNA strand-specific information, which is beneficial to RNA analysis such as gene annotation.

The present invention combines multiple reaction steps mentioned in the experimental principle into the same experimental operation step by optimizing the reaction conditions. For example, polyadenylation tailing and reverse transcription reaction are combined into one step, and the two end adapters of cDNA are connected separately and combined into one step, thereby reducing the operation steps and time of library preparation, and the experimental operation is simpler and faster.

The present invention uses a DNA ligase scheme to replace the RNA ligase scheme in the miRNA library preparation method to add a sequencing adapter sequence through experimental design, thereby improving the efficiency of connection, shortening the time required for connection, and reducing the cost.

The present invention adopts PCR amplification technology to amplify the library sequence, introduces the Barcode sequence for library identification and the structural sequence required for cyclization reaction and sequencing through PCR technology, and can realize the mixing of multiple library samples for sequencing together, thereby improving the detection throughput and reducing the detection cost.

The method of the present invention is applicable to high-throughput sequencing detection of various sample types such as tissue cell RNA and free RNA, and can be applied to RNA samples with low starting amounts at the ng level or even the pg level.

The present invention can be used for free RNA detection in the field of liquid biopsy. Currently, there is no dedicated RNA library construction kit for free RNA samples on the market. This method has broad application prospects. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (24)

A method for preparing various types of RNA libraries, characterized by comprising: adding polyA to the end of an RNA sample, performing reverse transcription and U base digestion, and preparing a cDNA library; The RNA sample contains at least one of total RNA, rRNA, mRNA, tRNA, miRNA and/or lncRNA. The preparation method according to claim 1 is characterized in that the preparation method specifically includes: DNA digestion, RNA end modification, polyA tailing, reverse transcription, U base digestion, cDNA end modification, denaturation, linker addition and library construction. The preparation method according to claim 2, characterized in that The DNA digestion and RNA end modification are performed in a first system; The reverse transcription is performed in a second system; The step of adding the polyA tail is performed in the first system or the second system. The preparation method according to claim 2 or 3 is characterized in that the cDNA end modification and denaturation are carried out in a third system. The preparation method according to claim 4 is characterized in that the third system also includes a reagent for U base digestion. The preparation method according to claim 3, characterized in that the step of adding the polyA tail is carried out in a first system, wherein: The first system includes: RNA sample, PolyA polymerase reaction buffer, ATP, BSA, DNase I, T4 polynucleotide kinase, PolyA polymerase, RNase inhibitor and nuclease-free water; The second system includes: the reaction product of the first system, reverse transcription primer, HiScript III reaction buffer, HiScript III reverse transcriptase, dNTP Mix, RNase inhibitor and nuclease-free water. The preparation method according to claim 3, characterized in that the step of adding the polyA tail is carried out in a second system, wherein: The first system comprises: RNA sample, DNase I reaction buffer, ATP, DNase I, T4 polynucleotide kinase and RNase inhibitor; The second system includes: the reaction product of the first system, HiScript III reaction buffer, BSA, PEG8000, dNTP Mix, reverse transcription primer, PolyA polymerase, RNase inhibitor, HiScript III reverse transcriptase and nuclease-free water. The preparation method according to claim 6 or 7, characterized in that the reverse transcription primer has the following nucleotide sequence: poly(T)n-UVNm; Where n represents the number of bases T, and m represents the number of bases N; n is an integer of 8 to 50, and m is an integer of 1 to 4 At least one T in the poly(T)n is replaced by U; V is selected from any one of base A, base C and base G; and N is selected from any one of base A, base T, base C and base G. The preparation method according to claim 8 is characterized in that the reverse transcription primer has a nucleotide sequence as shown in SEQ ID NO:1. The preparation method according to claim 4, characterized in that The third system includes: the reaction product after U base digestion, polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer at pH 8.0, ultra-thermostable single-stranded binding protein and nuclease-free water. The preparation method according to claim 5, characterized in that The third system includes: the reaction product of polyA tailing and reverse transcription, polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer at pH 8.0, ultra-thermostable single-stranded binding protein, uracil-specific excision reagent USER enzyme and nuclease-free water. The preparation method according to claim 2 is characterized in that, in the step of adding a linker, the linker includes a 5' end linker and a 3' end linker, the 5' end linker sequence has a nucleotide sequence as shown in SEQ ID NO 2 and SEQ ID NO 3, and the 3' end linker sequence has a nucleotide sequence as shown in SEQ ID NO 4 and SEQ ID NO 5. A method for sequencing multiple types of RNA libraries, characterized in that the cDNA library prepared by the preparation method according to any one of claims 1 to 12 is used as a sample for on-machine sequencing. The sequencing method according to claim 13 is characterized in that the cDNA library is sequenced after PCR amplification, purification, sample mixing, and single-stranded circularization. The sequencing method according to claim 14 is characterized in that the upstream primer of the PCR amplification has a nucleotide sequence as shown in SEQ ID NO 6, and the downstream primer of the PCR amplification has a nucleotide sequence as shown in SEQ ID NO 7. The library construction reagents are characterized by comprising reagent I, reagent II and reagent III; The reagent I comprises: DNase I reaction buffer, ATP, DNase I, T4 polynucleotide kinase, and RNase inhibitor; The reagent II includes: reverse transcription primer, HiScript III reaction buffer, HiScript III reverse transcriptase, dNTP Mix, and RNase inhibitor; The reagent III includes: polynucleotide kinase reaction buffer, T4 polynucleotide kinase, Tris buffer at pH 8.0, and ultra-thermostable single-chain binding protein. The construction reagent according to claim 16, characterized in that The reagent I also includes: PolyA polymerase reaction buffer, BSA, and PolyA polymerase. The construction reagent according to claim 16, characterized in that The reagent II also includes: BSA, PEG8000, and PolyA polymerase. The construction reagent according to claim 16 is characterized in that the reagent III also includes a U base excision reagent, and the U base excision reagent includes a uracil-specific excision reagent USER enzyme. The construction reagent according to claim 16 is characterized in that it also includes a linker adding reagent, which includes: Tris-Hcl buffer, sodium chloride, EDTA, linkers as shown in SEQ ID NO 2 to 5, ligation reaction buffer, T4 ligase and hexamminecobalt chloride. The construction reagent according to claim 16 is characterized in that it also includes a purification reagent, and the purification reagent includes Agencourt AMPure XP magnetic beads. The construction reagent according to claim 16 is characterized in that it also includes a PCR amplification reagent, which includes a PCR enzyme reaction solution, an upstream primer of the nucleotide sequence shown in SEQ ID NO 6, and a downstream primer of the nucleotide sequence shown in SEQ ID NO 7. The construction reagent according to any one of claims 16 to 22, further comprising an RNA extraction reagent. Use of the construction reagent according to any one of claims 16 to 23 in preparing multiple types of RNA libraries.