CN111662969A - Gene transcription region multi-variable region sequencing method - Google Patents
Gene transcription region multi-variable region sequencing method Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and discloses a method for sequencing multiple variable regions of a gene transcription region, which comprises the following steps: step S1: detecting a sample; step S2: constructing a library; step S3: purifying mRNA; step S4: synthesizing cDNA; step S5: end repair and linker attachment; step S6: processing a target transcript; step S7: preparing a reaction system; step S8: PCR reaction and product recovery: preparing a PCR reaction system, setting a reaction program, and amplifying a connecting product; step S9: library detection: using the range of insert of the detection library, and simultaneously quantifying the concentration of the library; step S10: and (4) accurately sequencing. The gene transcription region variable region sequencing method greatly reduces sequencing data amount through new generation high-throughput sequencing, can comprehensively and quickly obtain frequency information of a target transcript sequence of a specific tissue or organ of a certain species in a specific state, and is suitable for different library building kits.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for sequencing multiple variable regions of a gene transcription region.
Background
Transcriptome sequencing is the sum of all RNAs that a particular cell can transcribe in a functional state, mainly including mRNA and non-coding RNAs. Transcriptome research is the basis and starting point of gene function and structure research, almost all transcript sequence information of a specific tissue or organ of a certain species in a certain state can be comprehensively and quickly obtained through a new generation of high-throughput sequencing, and the transcriptome research is widely applied to the fields of basic research, clinical diagnosis, drug research and development and the like. The transcriptome is the sum of all RNAs transcribed from a particular tissue or cell at a certain developmental stage or functional state, and mainly comprises mRNA and non-coding RNA (ncRNA). Transcriptome studies can study gene functions and gene structures from the global level, reveal specific biological processes, and have been widely applied in the fields of plant candidate gene discovery, functional identification, genetic improvement, and the like. With the commercialization of a new generation of sequencing platform, RNA sequencing (RNA-Seq) technology has become one of the important means for transcriptomics research.
The technology utilizes a new generation of high-throughput sequencing platform to sequence genome cDNA, calculates the expression quantity of different mRNA by counting related Reads (small fragments of cDNA for sequencing), analyzes the structure and expression level of a transcript, discovers unknown transcripts and rare transcripts at the same time, accurately identifies variable shearing sites and coding sequence single nucleotide polymorphism, and provides the most comprehensive transcriptome information. The technical process of transcriptome sequencing mainly comprises sample preparation, library construction, DNA clustering amplification, high-throughput sequencing and data analysis, and compared with the traditional chip hybridization platform, the RNA-Seq technology has a plurality of unique advantages, the transcriptome sequencing can detect the whole transcription activity of any species without designing a probe aiming at a known sequence in advance, provides more accurate digital signals, higher detection throughput and wider detection range, and is a powerful tool for deeply researching the transcriptome at present, so that the method provides a gene transcription region multiple variable region sequencing method.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for sequencing a multiple variable region of a gene transcription region.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for sequencing multiple variable regions of a gene transcription region can carry out accurate qualitative and quantitative determination on a target transcription, carries out primer A design according to the sequence condition of the target region transcription region, and carries out an experiment after the design, and comprises the following steps:
step S1: sample detection: detecting the concentration, RIN value, 28S/18S and fragment size of total RNA;
step S2: constructing a library;
step S3: purification of mRNA: taking a certain amount of total RNA samples, performing temperature-adapted denaturation to open the secondary structure of the total RNA, and purifying to obtain mRNA;
step S4: and (3) cDNA synthesis: adding a pre-prepared first-strand synthesis reaction system into the mRNA obtained in the step S3, synthesizing first-strand cDNA on a PCR instrument according to a corresponding program, preparing a second-strand synthesis reaction system, reacting at a proper temperature for a certain time, and synthesizing second-strand cDNA;
step S5: end repair and linker attachment: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, repairing the end of the double-stranded cDNA, preparing a linker connection reaction system, and carrying out a moderate temperature reaction for a certain time to connect the linker and the cDNA;
step S6: target transcript processing: amplifying the sequence with the sequencing library adaptor obtained in the step S5; amplification was performed using primer a we designed and a 3' sequencing library adaptor.
Step S7: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, preparing a joint connection reaction system, carrying out a moderate temperature reaction for a certain time, and linking a 5' end sequencing library joint with an amplification product in S7;
step S8: PCR reaction and product recovery: preparing a PCR reaction system, setting a reaction program, and amplifying a connecting product;
step S9: library detection: using the range of insert of the detection library, and simultaneously quantifying the concentration of the library;
step S10: and (3) accurate sequencing: and (4) enriching a large number of sequences of the diversity region of the transcript, and carrying out accurate sequencing.
Preferably, in step S1, the concentration of total RNA, RIN value, 28S/18S and fragment size were determined by using an Agilent2100 Bioanalyzer.
Preferably, in step S3, total RNA is purified by using oligo-dT magnetic beads.
Preferably, in step S5, the added 5' sequencing primer is synthesized according to the species and the sequence characteristics of the target transcript by unique design when amplifying the target transcript.
Preferably, in step S6, when the double-stranded cDNA ends are repaired, A bases are added to the 3' -end.
Preferably, in step S8, the detection library is Agilent2100 Bioanalyzer.
Preferably, in step S8, the concentration of the library is quantified by using the ABI StepOneplus Real-Time PCR System.
Compared with the prior art, the invention provides a method for sequencing a gene transcription region multiple variable region, which has the following beneficial effects:
(1) transcriptome research is the basis and starting point of gene function and structure research, 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 through new-generation high-throughput sequencing, and the process is suitable for different library building kits;
(2) by sequentially carrying out sample detection, library construction, mRNA purification, fragmentation treatment, cDNA synthesis, end repair and joint connection, PCR reaction, product recovery and library detection accurate sequencing, a good library can be quickly constructed;
(3) specific primer amplification is carried out on the constructed library again to form a sequence enriched to a specific gene transcript, and then secondary library construction is carried out, so that a large number of sequences of the variable regions of the transcript are enriched, and accurate sequencing is carried out;
(4) because the forms of the transcripts of organisms are more, the difficulty of researching specific transcripts is more complex, if a common sequencing library is researched, the sequencing data volume is huge, the sequencing is carried out after directional enrichment is carried out by using the technology, the data volume is one thousandth of the original data volume, a good project can be reached, and the method is very significant for researching some important genes;
(5) on the basis of transcriptome sequencing, the transcript of the gene can be subjected to special quantitative sequencing, the scientific significance brought by the base change of the transcript is explained, and the method is subsequently applied to microbial fermentation guidance production;
the gene transcription region variable region sequencing method greatly reduces sequencing data amount through new generation high-throughput sequencing, can comprehensively and quickly obtain almost all transcript sequence information of a specific tissue or organ of a certain species in a specific state, and is suitable for different library building kits.
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FIG. 1 is a flow chart of a method for sequencing a multiple variable region of a gene transcription region according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1, a method for sequencing multiple variable regions of a gene transcription region, which can accurately determine the nature and quantity of a target transcript, designs a primer A according to the sequence condition of the target region transcript, and performs an experiment after the design, and comprises the following steps:
step S1: sample detection: detecting the concentration, RIN value, 28S/18S and fragment size of total RNA;
step S2: constructing a library;
step S3: purification of mRNA: taking a certain amount of total RNA samples, performing temperature-adapted denaturation to open the secondary structure of the total RNA, and purifying to obtain mRNA;
step S4: and (3) cDNA synthesis: adding a pre-prepared first-strand synthesis reaction system into the mRNA obtained in the step S3, synthesizing first-strand cDNA on a PCR instrument according to a corresponding program, preparing a second-strand synthesis reaction system, reacting at a proper temperature for a certain time, and synthesizing second-strand cDNA;
step S5: end repair and linker attachment: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, repairing the end of the double-stranded cDNA, preparing a linker connection reaction system, and carrying out a moderate temperature reaction for a certain time to connect the linker and the cDNA;
step S6: target transcript processing: amplifying the sequence with the sequencing library adaptor obtained in the step S5; amplification was performed using primer a we designed and a 3' sequencing library adaptor.
Step S7: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, preparing a joint connection reaction system, carrying out a moderate temperature reaction for a certain time, and linking a 5' end sequencing library joint with an amplification product in S7;
step S8: PCR reaction and product recovery: preparing a PCR reaction system, setting a reaction program, and amplifying a connecting product;
step S9: library detection: using the range of insert of the detection library, and simultaneously quantifying the concentration of the library;
step S10: and (3) accurate sequencing: and (4) enriching a large number of sequences of the diversity region of the transcript, and carrying out accurate sequencing.
In this example, in step S1, the concentration of total RNA, RIN value, 28S/18S and fragment size were measured by using an Agilent2100Bioanalyzer, and in step S3, total RNA was purified by using oligo-dT magnetic beads.
In this example, the 5 '-end sequencing primer added during amplification of the target transcript in step S5 was designed and synthesized uniquely according to the species and sequence characteristics of the target transcript, and the A base was added to the 3' -end during repair of the double-stranded cDNA in step S6.
In this example, Agilent2100Bioanalyzer was used for the detection of the library in step S8, and the concentration of the library was quantified by using the ABI StepOneplus Real-Time PCR System in step S8.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A method for sequencing a multiple variable region of a gene transcription region, comprising the steps of:
step S1: sample detection: detecting the concentration, RIN value, 28S/18S and fragment size of total RNA;
step S2: constructing a library;
step S3: purification of mRNA: taking a certain amount of total RNA samples, performing temperature-adapted denaturation to open the secondary structure of the total RNA, and purifying to obtain mRNA;
step S4: and (3) cDNA synthesis: adding a pre-prepared first-strand synthesis reaction system into the mRNA obtained in the step S3, synthesizing first-strand cDNA on a PCR instrument according to a corresponding program, preparing a second-strand synthesis reaction system, reacting at a proper temperature for a certain time, and synthesizing second-strand cDNA;
step S5: end repair and linker attachment: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, repairing the end of the double-stranded cDNA, preparing a linker connection reaction system, and carrying out a moderate temperature reaction for a certain time to connect the linker and the cDNA;
step S6: target transcript processing: amplifying the sequence with the sequencing library adaptor obtained in the step S5; amplification was performed using designed primer a and 3' sequencing library adaptors.
Step S7: preparing a reaction system, carrying out a moderate temperature reaction for a certain time, preparing a joint connection reaction system, carrying out a moderate temperature reaction for a certain time, and linking a 5' end sequencing library joint with an amplification product in S7;
step S8: PCR reaction and product recovery: preparing a PCR reaction system, setting a reaction program, and amplifying a connecting product;
step S9: library detection: using the range of insert of the detection library, and simultaneously quantifying the concentration of the library;
step S10: and (3) accurate sequencing: and (4) enriching a large number of sequences of the diversity region of the transcript, and carrying out accurate sequencing.
2. The method of claim 1, wherein in step S1, the concentration of total RNA, RIN value, 28S/18S and fragment size are determined by Agilent2100 Bioanalyzer.
3. The method of claim 1, wherein in step S3, total RNA is purified by using oligo-dT magnetic beads.
4. The method of claim 1, wherein in step S5, the 5' sequencing primer is designed and synthesized according to the species and the sequence characteristics of the target transcript.
5. The method of claim 1, wherein in step S6, when the double-stranded cDNA ends are repaired, A bases are added to the 3' -end.
6. The method of claim 1, wherein in step S8, Agilent2100Bioanalyzer is used as the detection library.
7. The method for sequencing the multiple variable regions of a gene transcript according to claim 1, wherein in step S8, the concentration of the library is quantified by using ABI StepOneplus Real-Time PCR System.
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Application publication date: 20200915 |