CN109385468B

CN109385468B - Kit and method for detecting strand-specific efficiency

Info

Publication number: CN109385468B
Application number: CN201710684356.5A
Authority: CN
Inventors: 宗亮; 伍梓靖; 姚秋林; 谢梅英
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2017-08-11
Filing date: 2017-08-11
Publication date: 2022-08-16
Anticipated expiration: 2037-08-11
Also published as: CN109385468A

Abstract

The invention discloses a reagent set and a method for detecting chain specificity efficiency. The invention discloses a reagent set for detecting chain specificity efficiency, which consists of RNA shown in SEQ ID NO. 1-6, and a method for detecting chain specificity efficiency, which comprises the following steps: adding the complete set of reagents of the invention into RNA to be subjected to library building, then constructing a chain specificity transcriptome library, then carrying out high-throughput sequencing, comparing sequencing data with sequences of the complete set of reagents, calculating the proportion of the number of sense comparison reads of the complete set of reagents to the total number of reads of the complete set of reagents, wherein the proportion is a chain specificity coefficient, and determining the chain specificity efficiency according to the chain specificity coefficient. The method and the reagent set for detecting the chain specificity efficiency can be used for indicating the credibility of the sequencing data of the chain specificity transcriptome of mRNA or non-coding RNA and the like and ensuring the reliability of the analysis result.

Description

Kit and method for detecting strand-specific efficiency

Technical Field

The invention relates to a reagent set and a method for detecting chain specificity efficiency in the field of biotechnology.

Background

With the explosive development of NGS (next generation sequencing technology), the transcriptome sequencing technology has become a major means for studying gene expression. Transcriptomes are the necessary links connecting genomic genetic information with biological functions, and are the basis and starting point for gene function and structure research. Through the new generation of high-throughput sequencing, almost all transcript sequence information of a specific tissue or organ of a certain species in a specific state can be comprehensively and quickly obtained, and the method is widely applied to the fields of basic research, clinical diagnosis, drug research and the like.

However, the conventional transcriptome cannot provide characteristic information of chain direction in a sequencing sequence and cannot truly reflect the transcription condition. Thus, strand-specific transcriptome sequencing techniques (ssRNA-Seq) have emerged. By retaining the directional information of the mRNA strands when constructing the sequencing library, analysis of the data after sequencing can determine whether the transcripts are from sense or antisense DNA strands. Compared with the common transcriptome sequencing, the method can more accurately count the number of transcripts and determine the structure of genes, can find more antisense transcripts, and is widely applied to the field range of researching gene structures, regulating gene expression and the like at present.

There are several methods available to achieve strand-specific library construction, including: an RNA linker ligation method, a terminal template conversion method, a dUTP method, and the like. Common methods reported in the literature for assessing strand-specific transcriptome database data are: species information of experimental samples is clarified, and a reference gene set is searched in an internet database, all genes of the experimental samples are assumed to have no antisense expression phenomenon, then the reading section ratio from antisense strands in sequencing data is counted, and the quality of strand-specific transcriptome data is evaluated according to the ratio.

Existing assessment schemes have several limitations in their application. First, with the expansion of the research range of transcriptome, there are a lot of experimental samples whose reference gene sequences are incomplete or with low confidence, which results in the failure of the existing evaluation schemes. Secondly, supposing that all genes do not have antisense expression phenomenon, the method is a very hard hypothesis, and samples in different species and different physiological states inevitably have antisense expression events of different degrees; the discovery of the actual presence of antisense expression events is also an experimental goal of strand-specific transcriptome sequencing. Thirdly, reverse transcriptase used in the process of building a strand-specific transcriptome library may have strong DDDP (DNA-Dependent DNA Polymerase activities) activity, differences in PCR enzymes used in template amplification steps, and other factors, and may cause the reduction of strand-specific efficiency, for example, the research on sequencing of the strand-specific transcriptome developed by Shenzhen Huada Gen Ltd, but the quantitative result identified by the RNA sample has low consistency with the QPCR verification experiment, and Pearson coefficient is only 0.542 (FIG. 1). Finally, the existing evaluation scheme needs to perform an additional antisense comparison operation in the bioinformatics process, which consumes much computing resources and time.

High-efficiency chain-specific processing is a prerequisite for ensuring accurate transcript quantification and gene structure optimization results, but a mature detection scheme for calculating the chain-specific processing efficiency in sequencing data does not exist at present. This severely limits the application and development of strand-specific transcriptome sequencing technologies.

Disclosure of Invention

The present invention provides, in a first aspect, a kit for detecting strand-specific efficiency.

The kit provided by the invention is at least one of RNAs respectively named SCS1, SCS2, SCS3, SCS4, SCS5 and SCS 6;

the SCS1 is any one of the following RNAs a1) to a 4):

a1) RNA shown as SEQ ID NO. 1 in the sequence list;

a2) RNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of a 1);

a3) RNA with more than 85% identity to RNA defined by a1) or a 2);

a4) RNA that hybridizes under stringent conditions to the RNA defined under a1) or a 2);

the SCS2 is any one of the following RNAs from b1) to b 4):

b1) RNA shown as SEQ ID NO. 2 in the sequence list;

b2) RNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);

b3) RNA having more than 85% identity to RNA defined by b1) or b 2);

b4) RNA that hybridizes under stringent conditions to the RNA defined in b1) or b 2);

the SCS3 is any one of the following RNAs from c1) to c 4):

c1) RNA shown as SEQ ID NO. 3 in the sequence list;

c2) RNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of c 1);

c3) RNA having more than 85% identity to RNA defined by c1) or c 2);

c4) RNA that hybridizes under stringent conditions to an RNA defined under c1) or c 2);

the SCS4 is any one of the following RNA from d1) to d 4):

d1) RNA shown as SEQ ID NO. 4 in the sequence list;

d2) RNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of d 1);

d3) RNA having more than 85% identity to RNA defined by d1) or d 2);

d4) RNA that hybridizes under stringent conditions to an RNA defined under d1) or d 2);

the SCS5 is any one of the following RNAs from e1) to e 4):

e1) RNA shown as SEQ ID NO. 5 in the sequence list;

e2) RNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of e 1);

e3) RNA having more than 85% identity to RNA defined by e1) or e 2);

e4) RNA that hybridizes under stringent conditions to an RNA defined in e1) or e 2);

the SCS6 is any one of the following RNA from f1) to f 4):

f1) RNA shown as SEQ ID NO. 6 in the sequence list;

f2) RNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of f 1);

f3) an RNA having 85% or more identity to the RNA defined by f1) or f 2);

f4) RNA that hybridizes under stringent conditions to the RNA defined in f1) or f 2).

The RNA may be single-stranded RNA or double-stranded RNA.

Wherein the addition of one or several nucleotides may be an addition of one to ten nucleotides.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences having 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence set forth in any one of SEQ ID NOs 1 to 6 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The stringent conditions are hybridization and washing of the membrane 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 85% or more may be 85%, 90% or 95% or more.

The invention also provides a method for detecting chain-specific efficiency, comprising:

1) adding exogenous RNA into RNA of a library to be built, wherein the exogenous RNA is inconsistent with an RNA sequence in an organism to obtain mixed RNA;

2) constructing a strand-specific transcriptome library for the mixed RNA to obtain a strand-specific transcriptome library;

3) performing high-throughput sequencing on the chain specificity transcriptome library to obtain sequencing data;

4) comparing the sequencing data with the sequence of the exogenous RNA, calculating the proportion of the number of positive sense comparison reads of the exogenous RNA to the total number of reads of the exogenous RNA, wherein the proportion is a chain specificity coefficient, and determining the chain specificity efficiency according to the chain specificity coefficient;

the sense alignment reads of the exogenous RNA are reads that have the same sequence as any RNA or any fragment thereof in the exogenous RNA.

In the above method, the exogenous RNA may be the kit.

In the above method, the higher the chain specificity coefficient is, the higher the chain specificity efficiency is, and the lower the chain specificity coefficient is, the lower the chain specificity efficiency is.

The construction of the strand-specific transcriptome library for the mixed RNA in the step 2) can be carried out by adopting a method in the prior art, and specifically, the method sequentially comprises the following steps: fragmenting the mixed RNA, carrying out reverse transcription to obtain a cDNA single strand, inserting dUTP to synthesize a cDNA double strand, repairing the end of the cDNA and connecting a joint, and amplifying the cDNA by a PCR method to obtain a strand specific transcriptome library.

In the above method, the mass ratio of the RNA to be pooled to the exogenous RNA may be (0.5-2). times.10 ⁴ ：1。

The mass ratio of the RNA of the library to be built to the exogenous RNA can be specifically 1 × 10 ⁴ :1。

In the above method, step 1) may further comprise fragmenting the RNA to be pooled before adding the exogenous RNA.

In the above method, step 1) may further include 11) and 12):

11) enriching mRNA in the RNA of the library to be built to obtain an mRNA library;

12) adding the exogenous RNA to the mRNA library to obtain a mixed RNA.

In the above method, the mass ratio of the mRNA in the mRNA library to the exogenous RNA may be 1% or less.

In the above method, step 12) may further comprise fragmenting the mRNA of the mRNA pool before adding the exogenous RNA.

The invention also provides a method for constructing a chain-specific transcriptome library, which comprises the following steps:

1) adding the exogenous RNA into RNA of a library to be built to obtain mixed RNA;

2) and constructing a strand-specific transcriptome library on the mixed RNA to obtain the strand-specific transcriptome library.

The invention also provides a method of strand-specific transcriptome sequencing, the method comprising: and constructing a chain specificity transcriptome library by the transcriptome to be sequenced according to the construction method of the chain specificity transcriptome library, and sequencing the obtained chain specificity transcriptome library to complete the sequencing of the transcriptome.

The invention also provides any one of the following applications of the kit:

x1, in the preparation of chain-specific efficiency products;

x2, use in chain-specific efficiency;

x3, in the preparation of chain-specific transcriptome library construction products;

x4, use in the construction of a chain-specific transcriptome library.

The invention utilizes the chain specificity efficiency to evaluate the construction efficiency of the chain specificity transcriptome library (namely the efficiency of chain specificity treatment), namely the reading ratio of a sense chain, so as to evaluate the quality of the chain specificity transcriptome data, and a method for detecting the chain specificity efficiency is established by designing a complete set of reagents for detecting the chain specificity efficiency. The chain specificity coefficient obtained by the method for detecting the chain specificity efficiency can directly reflect the real situation of the chain specificity efficiency in the experimental process. Verification results of QPCR show that the chain specificity efficiency and the change trend of the chain specificity coefficient have consistency, and the chain specificity coefficient can directly reflect the chain specificity efficiency, wherein the higher the chain specificity coefficient is, the higher the chain specificity efficiency is, and the lower the chain specificity coefficient is, the lower the chain specificity efficiency is. The method for detecting the chain specificity efficiency is an effective detection method, can be used for indicating the credibility of the sequencing data of the chain specificity transcriptome such as mRNA or non-coding RNA and the like, and ensures the reliability of the analysis result.

Drawings

FIG. 1 is a comparison of RNA sample quantification results with QPCR validation experiment consistency.

FIG. 2 is a flow chart for detecting strand-specific efficiency.

FIG. 3 is a flow chart of a method for using FLAG information to distinguish the results of a positive-negative alignment.

FIG. 4 shows the number of reads for sense and antisense alignments for different batches.

FIG. 5 is a correlation analysis of QPCR results with gene quantification results of the first batch of the strand specific transcriptome library.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 detection of chain-specific efficiency

This example provides a method for detecting strand-specific efficiency using a kit for detecting strand-specific efficiency, the kit for detecting strand-specific efficiency consisting of reagents named SCS1, SCS2, SCS3, SCS4, SCS5, and SCS6, respectively;

SCS1 is single-stranded RNA shown in SEQ ID NO. 1 in the sequence table;

SCS2 is single-stranded RNA shown in SEQ ID NO. 2 in the sequence table;

SCS3 is single-stranded RNA shown in SEQ ID NO. 3 in the sequence table;

SCS4 is single-stranded RNA shown in SEQ ID NO. 4 in the sequence table;

SCS5 is single-stranded RNA shown in SEQ ID NO. 5 in the sequence table;

SCS6 is single-stranded RNA shown in SEQ ID NO. 6 of the sequence list.

In the kit, the molar ratio of SCS1, SCS2, SCS3, SCS4, SCS5 and SCS6 can be 1:1:1:1: 1.

1. Detection of chain-specific efficiency

The specific method for detecting the chain-specific efficiency is as follows, and the flow chart is shown in the attached figure 2:

(1) determining the total RNA mass of the RNA sample, and estimating the mRNA mass according to the condition that the mRNA accounts for 1% of the total RNA;

(2) enriching and fragmenting mRNA, and then adding exogenous sequence SCS (single stranded nucleic acid) with the amount of 1% of the estimated amount of the mRNA, namely the reagent set for detecting the chain specificity efficiency into the fragmented mRNA to obtain mixed RNA;

(3) dividing the mixed RNA into three parts, and constructing a triple strand specific transcriptome library by using the same reagents and methods in the prior art to obtain three batches of strand specific transcriptome libraries, wherein all the reverse transcriptases are SuperScript ^TM II Reverse Transcriptase (Thermo Fisher Scientific); reading base information by adopting a high-throughput sequencing technology Illumina HiSeq4000 sequencing platform based on a sequencing while synthesizing technology to obtain three batches of original sequencing data of the RNA sample to be detected;

the process of constructing a chain-specific transcriptome library comprises: enriching mRNA, fragmenting, reverse transcribing to obtain cDNA strand, inserting dUTP to synthesize cDNA strand, repairing cDNA end and connecting joint, PCR amplifying cDNA to obtain library, and sequencing the library in HiSeq4000 machine.

(4) After the sequencing data are filtered, carrying out sequence comparison with an exogenous sequence SCS (exogenous transcript) to obtain an SAM file;

(5) by calculating and classifying FLAG (FLAG is a mark character used for representing the result after comparison with a reference sequence in a sequence file with SAM/BAM format) information, distinguishing sense comparison and antisense comparison, and referring to figure 3; the positive sense alignment reading is the reading which is the same as any RNA or any fragment sequence thereof in the exogenous sequence SCS; the antisense alignment reading segment is a reading segment which is complementary with any RNA or any fragment sequence thereof in the exogenous sequence SCS;

(6) counting the number of reads of sense comparison and antisense comparison in each batch respectively, see fig. 4, and calculating the ratio of the number of reads of sense comparison to the total number of reads, i.e. the chain Specificity Coefficient (SC), to obtain the data chain Specificity coefficients SC corresponding to 3 batches, which are 99.98%, 96.74% and 99.75%, respectively; because the strand specificity coefficient is the ratio of the number of the reads of the sense comparison of the reagent set to the total number of the reads, the relationship between the number of the reads of the sense comparison and the total number of the reads in the mixed RNA can be directly reflected, so that the strand specificity efficiency can be directly determined, the higher the strand specificity coefficient is, the higher the strand specificity efficiency is, and the lower the strand specificity coefficient is, the lower the strand specificity efficiency is;

(7) by comparing the experimental results of 3 batches, it is found that there is a more obvious abnormality in the chain specificity coefficient of the second batch, and the chain specificity coefficient of the first batch is the highest, indicating that the chain specificity efficiency of the first batch is the highest among the three batches.

2. QPCR validation of chain-specific efficiency

QPCR verification is carried out on the RNA samples of the three batches in the step 1, a standard sample UHRR (Agilent technologies) is researched by adopting a transcriptome sequencing methodology, QPCR experiments are carried out on about 1000 genes in the sample, the Expression quantity results are shared, and the results can be downloaded in a Gene Expression Omnibus website for retrieving GSE 5350.

The results were correlated with the gene quantification results of the three batches of the strand-specific transcriptome library of step one, respectively, and the results showed that the QPCR results and the gene quantification results of the first, second and third batches of the strand-specific transcriptome library were 0.809 (fig. 5), 0.611 and 0.814, respectively. It is shown that the chain specificity efficiencies of the first, third and second batches gradually decrease, consistent with the trend of the chain specificity coefficients, further indicating that the chain specificity coefficients can directly reflect the chain specificity efficiencies, with higher chain specificity coefficients being more efficient and lower chain specificity coefficients being less efficient.

<110> Shenzhen Hua Dagen shares GmbH

<120> reagent set and method for detecting strand-specific efficiency

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Claims

1. A kit for strand-specific efficiency detection in strand-specific transcriptome sequencing, consisting of RNAs named SCS1, SCS2, SCS3, SCS4, SCS5, and SCS6, respectively;

the SCS1 is RNA shown as SEQ ID NO. 1 in a sequence table;

the SCS2 is RNA shown as SEQ ID NO. 2 in a sequence table;

the SCS3 is RNA shown as SEQ ID NO. 3 in a sequence table;

the SCS4 is RNA shown as SEQ ID NO. 4 in a sequence table;

the SCS5 is RNA shown as SEQ ID NO. 5 in a sequence table;

the SCS6 is RNA shown as SEQ ID NO. 6 in a sequence table.

2. A method for detecting chain-specific efficiency, comprising:

1) adding exogenous RNA into RNA of a library to be built, wherein the exogenous RNA is inconsistent with an RNA sequence in an organism to obtain mixed RNA; the exogenous RNA is the kit of claim 1;

3. The method of claim 2, wherein: the mass ratio of the RNA of the library to be built to the exogenous RNA is (0.5-2) x 10 ⁴ ：1。

4. A method according to claim 2 or 3, characterized in that: step 1) further comprises fragmenting the RNA to be pooled before adding the exogenous RNA.

5. The method of claim 2, wherein: step 1) also includes 11) and 12):

12) adding the exogenous RNA to the mRNA library to obtain mixed RNA.

6. The method of claim 5, wherein: the mass ratio of the mRNA in the mRNA library to the exogenous RNA is less than or equal to 1%;

and/or, step 12) further comprises fragmenting mRNA in said mRNA pool prior to adding said exogenous RNA.

7. Use of the kit of claim 1 for any of the following:

x1, in the preparation of a chain-specific efficiency product in chain-specific transcriptome sequencing;

x2, chain specific efficiency in chain specific transcriptome sequencing.