CN113046415A

CN113046415A - Construction method and application of RNA sequencing library

Info

Publication number: CN113046415A
Application number: CN202110289299.7A
Authority: CN
Inventors: 盖伟; 边素莹
Original assignee: Micro Rock Medical Technology Beijing Co ltd
Current assignee: Micro Rock Medical Technology Beijing Co ltd
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2021-06-29

Abstract

The invention discloses a construction method of an RNA sequencing library, which is characterized by comprising a step of marking a target RNA sequence by a label before constructing the library, wherein the label is named as Rx and consists of two parts: the 5' end is a specific tag sequence Tx; the 3' end is a random sequence Nx; x in the tag Rx is a number, and a user can name according to requirements, such as R1, R2 and R3; the tag sequence Tx is a fixed nucleic acid sequence and consists of 5-18 bases, and a user can select the tag sequence as required but the tag sequence Tx is not identical to a target sequence; the random sequence Nx is composed of a random base sequence, N may be any one of A, T, C, G, and x represents the number of N-containing bases, and the number of bases may be 5 to 15. The invention also discloses application of the construction method of the RNA sequencing library. The construction method of the RNA sequencing library disclosed by the invention can effectively reduce the pollution of nucleic acid in the reagent, reduce background noise and simplify the bioinformatics analysis process.

Description

Construction method and application of RNA sequencing library

Technical Field

The invention relates to the technical field of high-throughput gene sequencing, in particular to a construction method and application of an RNA sequencing library.

Background

With the development of high-throughput sequencing technology (NGS), since 2004, the cost of high-throughput sequencing has been reduced by several orders of magnitude, which has led to its wide application in the fields of tumor detection, gene screening, etc. The successful application of the method in the field of pathogenic microorganism detection is firstly reported in 2014, and the method is widely applied in the field of clinical pathogenic detection in the following years, particularly in the field of limitation of a conventional detection method. The applications of NGS in clinical microbiological testing are diverse, including metagenomic NGS, i.e., mNGS, which can unbiased detect pathogens, can be used to identify pathogens directly from clinical specimens of patients without relying on traditional culture methods, and provide a reliable detection platform for pathogen detection that is difficult to culture or cannot be cultured in the laboratory.

Although the mNGS can detect microorganisms without hypothesis or bias and discover new microorganisms, it still has certain disadvantages, such as interference of host background nucleic acid in patient samples, and researches show that the sequences of pathogenic microorganisms for identification in clinical samples are relatively few, and the majority is host (> 99%) nucleic acid sequences, which brings great challenges to the application of the mNGS in pathogen detection. The host sequence can be subjected to host elimination in the stages of sample preparation and bioinformatics analysis, and a good effect on host elimination is achieved at present. A further disadvantage of the mNGS is that it detects the presence of background microbial contamination in the sample, including microbial contamination in reagents or laboratory environments used in extraction, banking, etc. Microbial contamination in a laboratory environment can be effectively ameliorated by decontaminating the laboratory environment. However, the contamination from the detection reagent is difficult to remove by conventional means, which brings great difficulty to the subsequent sequencing data analysis.

Currently, NGS is widely used in RNA sequencing, and it is usually necessary to construct an RNA sequencing library in the RNA sequencing process, and the prior art generally comprises the following steps: breaking the RNA fragment to a certain length by a mechanical method or an enzymatic method, purifying the broken RNA, performing reverse transcription by using a random primer, synthesizing a cDNA double strand, repairing the tail end, adding A and a joint, purifying, performing index PCR amplification and purifying an index PCR product. The library building method comprises three complicated purification steps, and great waste is caused to experimental reagents, consumables, time and manpower. Meanwhile, random primers are needed in the library building process, and sometimes the random primers bring imbalance of reverse transcription and bring certain deviation to a sequencing result.

Therefore, it is necessary to develop a novel RNA sequencing library construction method and reasonably utilize the method, and the method has great significance for improving the quality and efficiency of RNA library construction and solving or reducing the pollution of microbial nucleic acid in reagents to pathogen detection.

Disclosure of Invention

The invention mainly aims to provide a construction method of an RNA sequencing library and application thereof, wherein the construction method effectively reduces noise pollution of background microbial nucleic acid in a kit by adding a section of label mark to an RNA template before library construction, optimizes a biological signal analysis process and improves the sensitivity of RNA sequencing detection; the method can mark the RNA template, can effectively retain related information of the RNA template, converts the RNA template into DNA for library construction, and simultaneously converts the RNA template into small fragments which can be used for library construction and have the length of 50-1000 bp; the nucleic acid marked by the technology is constructed with a library together with a DNA template, and the two groups of nucleic acids can be effectively distinguished by bioinformatics analysis.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: a method for constructing an RNA sequencing library, which is characterized by comprising the step of labeling a target RNA sequence by a label before constructing the library, wherein the label is named as Rx and consists of two parts: the 5' end is a specific tag sequence Tx; the 3' end is a random sequence Nx; x in the tag Rx is a number, and a user can name according to requirements, such as R1, R2 and R3 … …; the tag sequence Tx is a fixed nucleic acid sequence and consists of 5-18 bases, and a user can select the tag sequence as required but the tag sequence Tx is not identical to a target sequence; the random sequence Nx is composed of a random base sequence, N may be any one of A, T, C, G, and x represents the number of N-containing bases, and the number of bases may be 5 to 15.

Further, the step of labeling the target RNA sequence with a tag specifically comprises:

step S1, labeling the RNA template with the label sequence Tx, wherein the base sequence is fixed, and the template can be screened and distinguished according to the sequence during data analysis, so as to remove the background noise in the reagent;

step S2, combining the random sequence Nx with a complementary region on the template to form an RNA-DNA double-stranded structure, wherein the structure can be recognized by reverse transcriptase and combined with the reverse transcriptase to generate a first strand of cDNA, and the 5' end of the cDNA strand is generated and marked with a tag Tx;

step S3, when the random sequence Nx in Rx is combined with the complementary region in the synthesized first strand cDNA, it can form cDNA-cDNA double-chain structure with the first strand cDNA, the structure can be identified by DNA polymerase, and combined with it to synthesize the second strand cDNA, the second strand cDNA is synthesized, the 5 'end and 3' end both contain fixed base label sequence, 5 'end is Rx, 3' end is the reverse complementary sequence of Rx, the Rx marked sequence is generated.

Further, the sequence of the Rx label in the step S3 is a part of the RNA template, and the length of the Rx label is 50-1000 bp.

Further, the method for constructing the RNA sequencing library can also break long RNA into short RNA sequences, namely, fragmenting the RNA.

Further, the method for constructing the RNA sequencing library also comprises the step of constructing the library by directly using the nucleic acid sequence formed by the method according to the library construction process of the corresponding sequencing platform, or the step of mixing the nucleic acid sequence formed by the method with the fragmented DNA prepared from the same sample, and then constructing the library according to the library construction process of the corresponding sequencing platform.

Preferably, the sequencing platform is one of Illumina platform and Ion Torrent platform, but not limited to the two sequencing platforms.

Further, the application of the construction method of the RNA sequencing library is that the nucleic acid sequence formed by the method is directly constructed according to the library construction process of a corresponding sequencing platform, and the data analysis process comprises the following steps:

(1) performing quality evaluation on sequencing data, and removing low-quality reads such as short sequences with low quality and length less than 50bp, joints and the like;

(2) screening the data for sequences that are 5 'Tx-containing or 3' reverse complements of Tx;

(3) comparing the sequence screened in the previous step with a host genome sequence, and removing a sequence which can be matched with a host genome, wherein the host is a human genome;

(4) comparing and analyzing the screened data with a corresponding database to determine the species to which the screened data belongs;

(5) and generating a corresponding detection report.

Further, the application of the construction method of the RNA sequencing library is that the nucleic acid sequence formed by the method is mixed with the fragmented DNA prepared by the same sample, and then the library construction is carried out according to the library construction process of a corresponding sequencing platform, wherein the data analysis process comprises the following steps:

step C1: performing quality evaluation on sequencing data, and removing low-quality reads such as short sequences with low quality and length less than 50bp, joints and the like;

step C2: dividing sequences containing Tx at the 5 'end or sequences containing reverse complementary sequences of Tx at the 3' end in data meeting the requirements into a group, and marking the group as an RNA group; sequences without Tx at the 5 'end and reverse complementary sequences without Tx at the 3' end are divided into a group and are marked as a DNA group;

step C3: comparing the RNA group data with a host human genome sequence, and removing sequences which can be matched with the host genome;

step C4: comparing the residual data of the RNA group screened in the previous step with a pathogen genome database for analysis to determine the RNA pathogens contained in the residual data;

step C5: comparing the DNA group data with a host human genome sequence, and removing sequences which can be matched with the host genome;

step C6: comparing the residual data of the DNA group screened in the previous step with a pathogen genome database for analysis to determine the DNA pathogens contained in the residual data;

step C7: determining pathogens contained in the sample according to the analysis results of the RNA group and the DNA group;

step C8: and generating a corresponding detection report.

Further, the tag sequence Rx is R1, consisting of T8 and N6; the R1 nucleic acid sequence is 5 '-CAGATATCNNNNNN-3'; the T8 used contains 8 fixed bases, and the sequence is CAGATATC; n6 contains 6 random bases and has the sequence nnnnnnnn.

Further, the first strand synthesis method of cDNA comprises the following steps: uniformly mixing target RNA to be subjected to library construction with a label R1, wherein the final concentration of R1 is 2 mu M, and adding an RNase inhibitor, DTT, dNTP, AMV reverse transcriptase and corresponding Buffer thereof, wherein the concentrations of the components are respectively as follows: RNase inhibitor 1U/. mu.L, AMV reverse transcriptase 0.05-0.5U/. mu.L (preferably 0.1U/. mu.L), DTT 5mM, dNTP 1mM, mixed well, the reaction tube was placed in a PCR instrument and the following procedure was run: 10min at 25 ℃, 10-60min (preferably 30min) at 42 ℃, 5min at 70 ℃ and hold at 4 ℃.

Further, the second strand synthesis method of cDNA comprises the following steps: after the reaction procedure is completed, the reaction tube is taken out, the Klenow large fragment (3 '→ 5' exo-) with the final concentration of 0.05-0.5U/. mu.L (preferably 0.1U/. mu.L) is added and mixed uniformly, the reaction tube is placed in a PCR instrument, and the following procedures are carried out: 10-60min (preferably 30min) at 25 ℃, 5min at 75 ℃ and hold at 4 ℃.

Further, the method for constructing the RNA sequencing library further comprises the step of purifying a reaction product by using 1.8 times of magnetic beads after the reaction is finished, wherein the purified product is directly used for constructing the library or is mixed with the fragmented DNA nucleic acid for constructing the library.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) according to the method for constructing the RNA library, the RNA template is added with a section of label mark before the library is constructed, so that the noise pollution of background microbial nucleic acid in the kit can be effectively reduced, and the biogenesis analysis process is optimized.

(2) The method for constructing the RNA library not only can mark the RNA template, but also can effectively retain the related information of the RNA template, convert the RNA template into DNA for constructing the library, and simultaneously convert the RNA template into small fragments which can be used for constructing the library and have the length of 50-1000 bp.

(3) According to the method for constructing the RNA library, the marked nucleic acid and the DNA template are used together for constructing the library, and the two groups of nucleic acids can be effectively distinguished through bioinformatics analysis. The technology can obviously reduce the background noise of the kit, can effectively improve the sensitivity of RNA sequencing detection, and can be used for the research of RNA virus sequencing detection.

(4) The method for constructing the RNA library can effectively reduce the pollution of reagent nucleic acid in the construction process of the RNA library, improve the detection sensitivity of RNA, and can fragment the RNA template and be directly used for the subsequent library construction.

(5) The method for constructing the RNA library can also be used for simultaneously detecting the RNA pathogen and the DNA pathogen nucleic acid, can effectively detect the RNA pathogen and the DNA pathogen of a sample, improves the detection sensitivity and specificity of the RNA pathogen, and is suitable for the field of pathogen detection.

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The following examples relate to apparatus comprising: clean bench, centrifuge, qubit4.0, PCR appearance, pipettor, magnetic frame, sequencer etc..

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

This example discloses a method for RNA library construction, comprising the steps of:

reverse transcription of RNA into cDNA

(1) Dividing the extracted nucleic acid of the respiratory syncytial virus into 2 parts, each 13 mu L, adding 4 mu L of 5 XBuffer into the first part, only containing random primers of the N6 sequence (the final concentration is 2 mu M), dNTP (the final concentration is 1mM), DTT (the final concentration is 5mM), RNase inhibitor (the final concentration is 1U/mu L), AMV reverse transcriptase (the final concentration is 0.1U/mu L), and supplementing 20 mu L of nuclease-free water; mu.L of 5 XBuffer, tag sequence R1 (final concentration 2. mu.M), dNTP (final concentration 1mM), DTT (final concentration 5mM), RNase inhibitor (final concentration 1U/. mu.L), AMV reverse transcriptase (final concentration 0.1U/. mu.L) was added to the second nucleic acid, and 20. mu.L of nuclease-free water was added.

(2) Mixing the prepared 2 parts of system in a vortex, performing instantaneous centrifugation, and placing the mixture in a PCR instrument for reaction; the reaction program is that the temperature is 25 ℃ for 5 min; 30min at 42 ℃; 5min at 70 ℃; hold at 4 ℃;

(3) after the reaction is finished, adding 1 mu LKLenow enzyme into a reaction system, uniformly mixing, performing instantaneous centrifugation, and placing in a PCR instrument for reaction; the reaction procedure is as follows: 30min at 25 ℃; 5min at 75 ℃; hold at 4 DEG C

(4) After the reaction, the reaction mixture was purified with 1.8X AMPure XP magnetic beads to obtain a virus nucleic acid after reverse transcription.

(II) library construction

The Ion Proton sequencing platform adopted by the invention adopts an Ion Proton platform library building process. The reagent Kit is prepared by the operation flow of an Ion Xpress Fragment Library Kit and an Ion Xpress Barcode Adapters 1-16 Kit according to the operation instruction.

(1) Taking 12.5 mu L of the reverse-transcribed nucleic acid, and sequentially adding 24.5 mu L of nuclease-free water, 5 mu L of ligase Buffer, 1 mu L of dNTP, 1 mu L of LDNA ligase, 4 mu L of shell-lacking repair enzyme, 1 mu L of universal linker and 1 mu L of linker containing Barcode X (different Barcode is adopted for each sample); mixing evenly, performing instantaneous centrifugation, placing the reaction tube in a PCR instrument, and performing reaction procedures: at 25 ℃ for 20 min; 72 ℃ for 5 min; hold at 4 ℃.

(2) After the reaction is finished, purifying by using 1.5 multiplied by AMPure XP magnetic beads, eluting nucleic acid by using 14 mu LTE, taking 12.5 mu L of nucleic acid solution added with a joint, adding 50 mu L of PCR mixed solution and 2.5 mu L of amplification primers; mixing evenly, centrifuging instantaneously, and placing the reaction tube in a PCR instrument; reaction procedure: pre-denaturation at 95 deg.C for 5 min; circulating phase (10 cycles) at 95 deg.C for 15s, 58 deg.C for 15s, and 70 deg.C for 1 min; 4 ℃, Hold;

(3) after completion of the reaction, the reaction mixture was purified with 1.5 × AMPure XP magnetic beads, and the nucleic acid was eluted with 20 μ L of TE.

(4) Library mixing, library template preparation and on-machine sequencing are carried out according to requirements.

(III) data analysis and result comparison

1. Analysis of sample sequencing data by reverse transcription Using tag R1

And performing quality evaluation on the sequencing data, and removing low-quality reads such as segment sequences with low quality and length less than 50bp, joints and the like. The 5' in the screening data contained T8: the sequence of CAGATATC or the sequence 3' of GATATCTG, which contains the reverse complement of T8. And comparing the data screened in the previous step with the host human genome sequence, and removing the sequence which can be matched with the host genome. And comparing the screened data with a microbial pathogen database for analysis, and determining the species to which the screened data belong. And generating a corresponding detection report.

2. Data analysis of reverse transcription samples Using N6 primer

And performing quality evaluation on the sequencing data, and removing low-quality reads such as segment sequences with low quality and length less than 50bp, joints and the like. And comparing the data screened in the previous step with the host human genome sequence, and removing the sequence which can be matched with the host genome. And comparing the screened data with a microbial pathogen database for analysis, and determining the species to which the screened data belong. And generating a corresponding detection report.

3. And (5) comparing detection results.

The ratio of each data in the sequencing data is shown in table 1 below. In the case of comparable data throughput, the background noise sequence of the final output data obtained by cDNA synthesis using the tag R1 of the present invention was only 0.9% of the data used for the final output data, whereas the background noise sequence of the final output data obtained by cDNA synthesis using conventional primers was 7.7% of the data used for the final output data, and the background noise was reduced by nearly 90% (88.2%) using the method of the present invention. The number of the nucleic acid reads of the respiratory syncytial virus detected by the method is obviously more than that of 123287Vs 46389 detected by the conventional method. It is demonstrated that the use of this technique can effectively reduce the background nucleic acid in the reagent and improve the detection sensitivity of RNA virus.

TABLE 1 comparison of data from the present invention and general RNA library construction methods

Example 2

This example discloses a method for co-sequencing RNA pathogen nucleic acid and DNA pathogen nucleic acid, comprising the steps of:

(I) sample Source and nucleic acid extraction

1. Sample source: clinically confirmed RNA virus, DNA virus and bacteria co-infected alveolar lavage fluid samples in 3 cases.

2. A300. mu.L sample was centrifuged at 12000rpm for 2min, and the supernatant was extracted with a viral nucleic acid extraction kit from QIAGEN. The operation flow is carried out according to the operation instruction.

3. And extracting the bacterial genome nucleic acid from the residual precipitate by using a Jinmaige bacterial genome nucleic acid extraction kit. The operation flow is carried out according to the extraction instruction.

(II) cDNA Synthesis of nucleic acids extracted from RNA pathogens

13. mu.L of each sample of viral nucleic acid was used for cDNA synthesis using the tag R1 in accordance with example 1, and 13. mu.L of viral nucleic acid was used for cDNA synthesis using N6 primer in accordance with example 1.

(III) Co-banking of RNA and DNA pathogen nucleic acids and on-machine sequencing

1. DNA nucleic acid fragmentation 20. mu.L of extracted viral nucleic acid and 20. mu.L of extracted bacterial nucleic acid were mixed and then subjected to nucleic acid fragmentation using Kapa fragmentation kit. The specific operation flow is as follows: taking 40 mu L of mixed nucleic acid, and adding 5 mu L of fragmentation enzyme and 5 mu L of fragmentation Buffer; mixing evenly, centrifuging instantaneously, and placing the reaction tube in a PCR instrument for reaction; the reaction procedure is as follows: 4 ℃, 1 min; at 37 ℃ for 40 min; 4 ℃, Hold; after the reaction is finished, adding 5 mu L of termination buffer solution, mixing uniformly in a vortex mode, and centrifuging instantaneously; purifying with 1.8 × AMPure XP magnetic bead; elution of nucleic acids with nuclease-free water

2. Separately, cDNA synthesized by R1 and fragmented DNA nucleic acid were mixed in equal volumes, while cDNA synthesized by N6 and fragmented DNA nucleic acid were mixed in equal volumes. Then, two sets of nucleic acids of each sample were subjected to Library construction using an Ion Xpress Fragment Library Kit and an Ion Xpress BarcodeAdapters 1-16 Kit, and the procedures were carried out with reference to example 1.

3. And performing library mixing, template preparation and on-machine sequencing on the constructed library according to requirements.

(IV) data analysis

1. Bioinformatic analysis of sequencing data containing tag R1:

(2) the data meeting the requirements contain T8 according to 5': the sequence of CAGATATC or the sequence of a reverse complementary sequence GATATCTG containing Tx at the 3' is divided into a group and is marked as an RNA group; the 5' end does not contain T8: the sequences of CAGATATC and 3' of reverse complementary sequences without Tx are divided into a group, and the group is marked as a DNA group; comparing the RNA group data with a host human genome sequence, and removing sequences which can be matched with the host genome;

(3) comparing and analyzing the RNA group residual data pathogen genome database screened in the previous step to determine RNA pathogen genes contained in the RNA group residual data pathogen genome database; comparing the DNA group data with a host human genome sequence, and removing sequences which can be matched with the host genome; comparing the residual data of the DNA group screened in the previous step with a pathogen genome database for analysis to determine DNA pathogen genes contained in the residual data; determining pathogens contained in the sample according to the analysis results of the RNA group and the DNA group; and generating a corresponding detection report.

2. Data analysis of reverse transcription samples Using N6 primer

(1) And performing quality evaluation on the sequencing data, and removing low-quality reads such as segment sequences with low quality and length less than 50bp, joints and the like.

(2) And comparing the data screened in the previous step with the host human genome sequence, and removing the sequence which can be matched with the host genome.

(3) And comparing the screened data with a microbial pathogen database for analysis, and determining the species to which the screened data belong.

(4) And generating a corresponding detection report.

3. Analysis of detection results

The detection results of 3 samples for sequencing are shown in tables 2, 3 and 4, and the detection results in the tables show that the RNA library construction method can effectively reduce the background noise of the co-constructed library, the noise is reduced by 80-90%, and the method has positive influence on data analysis. And the detection effect of RNA virus in the co-infection sample is obviously better than that of the traditional RNA library construction technology, and the detection amount of RNA is improved by 30-150%. And has no obvious influence on the detection of DNA. Therefore, the RNA database building technology can be used for the co-sequencing detection of RNA pathogens and DNA pathogens.

TABLE 2 sample BALF001 assay results

TABLE 3 sample BALF002 test results

TABLE 4 sample BALF003 test results

The foregoing is directed to embodiments of the present invention and, more particularly, to a method and apparatus for controlling a power converter in a power converter, including a power converter, a power.

Claims

1. A method for constructing an RNA sequencing library, which is characterized by comprising the step of labeling a target RNA sequence by a label before constructing the library, wherein the label is named as Rx and consists of two parts: the 5' end is a specific tag sequence Tx; the 3' end is a random sequence Nx; x in the tag Rx is a number, and the user can name the tag according to the requirement, such as R1, R2, R3 … …; the tag sequence Tx is a fixed nucleic acid sequence and consists of 5-18 bases, and a user can select the tag sequence as required but the tag sequence Tx is not identical to a target sequence; the random sequence Nx is composed of a random nucleic acid sequence, N can be any one of A, T, C, G, x represents the number of N, and the number of bases can be 5-15.

2. The method for constructing an RNA sequencing library according to claim 1, wherein the step of labeling the target RNA sequence with a tag comprises:

step S1, labeling the RNA template with the label sequence Tx, wherein the base sequence is fixed, the template can be screened and distinguished according to the sequence during data analysis, and the background noise in the reagent is removed;

3. The method of claim 2, wherein the sequence of the Rx tag in step S3 is a portion of the RNA template and has a length of 50-1000 bp.

4. The method of claim 1, further comprising the steps of claim 2, wherein the method of constructing the RNA sequencing library can break long RNA fragments into short RNA fragments, i.e. fragmenting RNA; the construction method of the RNA sequencing library also comprises the step that the nucleic acid sequence formed by the method can be directly constructed according to the corresponding sequencing platform library construction process, or is mixed with fragmented DNA in the same sample and then constructed according to the corresponding sequencing platform library construction process.

5. The method for constructing the RNA sequencing library of claim 4, wherein the sequencing platform is selected from the group consisting of but not limited to an Illumina platform and an Ion Torrent platform.

6. The method for constructing an RNA sequencing library according to any one of claims 1 to 5, wherein the method for constructing an RNA sequencing library is applied to directly library nucleic acids formed by the method according to a library construction process of a corresponding sequencing platform, and a data analysis process comprises the following steps:

(5) and generating a corresponding detection report.

7. The method for constructing an RNA sequencing library according to any one of claims 1 to 5, wherein the method for constructing an RNA sequencing library is applied to the library construction according to a corresponding sequencing platform library construction process after mixing nucleic acids formed by the method with fragmented DNA in the same sample, and the data analysis process comprises the following steps:

step C8: and generating a corresponding detection report.

8. The method for constructing the RNA sequencing library of claim 2, wherein the tag sequence Rx is R1 and consists of T8 and N6; the R1 nucleic acid sequence is 5 '-CAGATATCNNNNNN-3'; the T8 used contains 8 fixed bases, and the sequence is CAGATATC; n6 contains 6 random bases and has the sequence nnnnnnnn.

9. The method of claim 2, wherein the first strand synthesis of cDNA comprises the steps of: uniformly mixing target RNA to be subjected to library construction with a label R1, wherein the final concentration of R1 is 2 mu M, and adding an RNase inhibitor, DTT, dNTP, AMV reverse transcriptase and corresponding Buffer thereof, wherein the concentrations of the components are respectively as follows: RNase inhibitor 1U/. mu.L, AMV reverse transcriptase 0.05-0.5U/. mu.L (preferably 0.1U/. mu.L), DTT 5mM, dNTP 1mM, mixed well, the reaction tube was placed in a PCR instrument and the following procedure was run: 10min at 25 ℃, 10-60min (preferably 30min) at 42 ℃, 5min at 70 ℃ and hold at 4 ℃; the second strand synthesis method of cDNA comprises the following steps: after the reaction procedure is completed, the reaction tube is taken out, the Klenow large fragment (3 '→ 5' exo-) with the final concentration of 0.05-0.5U/. mu.L (preferably 0.1U/. mu.L) is added and mixed uniformly, the reaction tube is placed in a PCR instrument, and the following procedures are carried out: 10-60min (preferably 30min) at 25 ℃, 5min at 75 ℃ and hold at 4 ℃.

10. The method of claim 1, further comprising purifying the reaction product with 1.8 x magnetic beads after the reaction is completed, wherein the purified product is used directly for library construction or mixed with fragmented DNA nucleic acids for library construction.