CN114134206B - FFPE sample RNA library and construction method thereof - Google Patents

FFPE sample RNA library and construction method thereof Download PDF

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CN114134206B
CN114134206B CN202111476559.8A CN202111476559A CN114134206B CN 114134206 B CN114134206 B CN 114134206B CN 202111476559 A CN202111476559 A CN 202111476559A CN 114134206 B CN114134206 B CN 114134206B
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amplification
library
primer
seq
rna
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CN114134206A (en
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李凤明
魏丽
许青
杜波
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Wuhan Zhenhe Medical Laboratory Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention relates to the technical field of biology, in particular to a construction method of an FFPE sample RNA fusion mutation library. In the method, a section of artificial sequence (plasmid) is added to carry out effective amplification and library establishment together with a primer pool panel for detecting RNA fusion mutation in the library establishment process, so that the probability of nonspecific collision, combination and amplification of panel primers is reduced and the generation of primers and adapter dimers is reduced due to the dominant amplification of the plasmid while the library output is ensured.

Description

FFPE sample RNA library and construction method thereof
Technical Field
The invention relates to the technical field of biology, in particular to an FFPE sample RNA library and a construction method thereof.
Background
In-vitro tumor tissues easily lose the original normal structure and function, and samples (FFPE) treated by Formalin-Fixed andParrffin-Embedded can be preserved for a long time at normal temperature, so that paraffin embedding preservation of the tumor tissues becomes the most common sample storage means in hospitals. The formalin is easy to degrade nucleic acid in tissues and crosslink molecules to different degrees, the high-temperature infiltration process of paraffin further accelerates the degradation of nucleic acid, the preservation time and environment have great influence on nucleic acid in samples, high-quality nucleic acid is extracted from paraffin sections, and a library which has complete information and can be used for subsequent RNA research is constructed, so that the method is a quite difficult problem.
Complexity of RNAFFPE samples: 1. cross-linking with proteins and other cellular components; 2. over time, formalin can be oxidized to formic acid, thereby causing base depurination and nucleic acid strand cleavage, resulting in lower extraction and RNA quality; 3. is easy to be polluted by the PCR inhibitor, and the sample contains the PCR inhibitor. The complexity of the method leads to difficult construction of RNA library based on amplicon methodology library construction and sequencing, and the library output is low, so that the library construction success rate is low; primer and linker dimers are severe and thus the library quality is poor.
The IlluminaMiniseq and Miseq sequencing platform has the minimum requirement of 2nM on the denaturation of the on-machine library, the denaturation effect is poor below 2nM, and the sequencing success rate is low; therefore, the library which can be used for sequencing the on-machine library is required to have a certain ex-warehouse concentration (for example, the Panel requirement RNA library ex-warehouse concentration is more than or equal to 0.4 ng/. Mu.l); in addition, due to the complexity of RNA samples, too high primer dimers and adaptor dimers are easily introduced during the pooling of FFPE RNA samples of low quality. Therefore, in clinical application, the conventional amplicon library construction method has low library quality and low library construction success rate. In order to solve the problems of low amplicon library quality (increasing the target fragment ratio of the library, reducing the non-main peak fragment ratio of primer, adaptor dimer and the like) and RNA library establishment success rate (lower RNA library outlet concentration), technological workers propose methods such as increasing the amplification cycle number, improving the sequencing depth and the like.
Defects and deficiencies of the prior art:
1. increasing the amplification cycle number can improve the success rate of RNA library construction, but the primer dimer (dimer) and the linker dimer which are too high are easily introduced in the low-quality FFPE RNA sample library construction process, and the too high linker dimer can cause inaccuracy of the main peak quantification of the library by adopting the method of qubit quantification, so that the accurate pooling of the library is affected.
2. The sequencing depth is increased, the single sample detection cost is directly increased, unnecessary waste is caused, and the FFPE sample with poor quality cannot be sequenced by the illuminea platform due to low ex-warehouse concentration.
Disclosure of Invention
In view of this, the present invention provides a method for constructing an FFPE sample RNA library. In the method, a section of artificial sequence (DNA fragment shown in SEQ ID NO: 1) and a primer pool panel mixed with upstream and downstream primers of a plasmid and capable of detecting RNA fusion mutation are added in the library construction process to effectively amplify and construct libraries, so that the library construction success rate and the quality of RNA amplicon libraries are improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a construction method of an FFPE sample RNA library, which comprises the following steps:
step 1: extracting RNA of the FFPE sample, and obtaining cDNA product through reverse transcription;
step 2: adding to the amplification system of the cDNA product a nucleic acid sequence comprising SEQ ID NO:1 and a plasmid for amplifying the DNA fragment shown in SEQ ID NO:1, a primer of the DNA fragment shown in the specification; the library was constructed according to the method of construction of RNA sequencing library.
In the process of library establishment, a section of DNA fragment artificial sequence is doped, namely the DNA fragment artificial sequence comprises SEQ ID NO:1, and a plasmid mixed with the fragment shown in SEQ ID NO: 2-3 the primer pool panel of the upstream and downstream primers which can be used for detecting RNA fusion mutation together perform effective amplification and library establishment. The DNA fragment has NO homologous sequence with human genome, can be artificially synthesized, and can be synthesized by taking spikey-09 plasmid as a template through the sequence of SEQ ID NO: 2-3. The sequences are as follows:
SEQ ID No.1：
TGTGAACTGTCATCGGTCCGATCAATTAGTCTAGTGTGCGTTATTCAGATCGAGTGAGTACATGATTCGTCAGTGTGGATCAATTACAGTTAGGCCGCTGACACATTAGTAACGTCGGCAAGCACTTAGTCGTGTCGTAAGCCAGTGTGTCGTGTCT。
upstream primer 1:
5’-CCTACACGACGCTCTTCCGATCTTGTGAACTGTCATCGGTCCG-3’
(SEQ ID No.2)；
downstream primer 2:
5’-TTCAGACGTGTGCTCTTCCGAAAGACACGACACACTGGCTT-3’(SEQ ID No.3)。
the sequence of the 5' end underline mark in the upstream and downstream primers is a tail sequence, and the tail sequence can be combined with an index primer sequence in the second round of PCR to form a complete joint for completing sequencing.
In some embodiments, the DNA product amplification system incorporates a spikey-09 plasmid with a copy number of 4.5X10-5.
Step 1, the method further comprises the step of preprocessing RNA before reverse transcription; the pretreatment is as follows: mixing the RNA with the reverse transcription mixed solution, and reacting for 5min at 65 ℃ to obtain a pretreatment reaction product.
In some embodiments, in step 2, the method of constructing an RNA sequencing library comprises: for said addition of SEQ ID NO:1, performing a first round of PCR amplification by using the DNA fragment amplification system, purifying and digesting, recovering a digested product, performing a second round of PCR amplification, and purifying to obtain an FFPE sample RNA library.
In some embodiments, in step 2, the amplification system of the first round of PCR amplification comprises:
1 μl of spikein plasmid, 4 μl of amplification mix 1,5 nM-10 nM primer pool for detecting RNA fusion mutation, 5 nM-10 nM SEQ ID NO: 2-3, 10. Mu.l-30 cDNA product, no nuclease water up to 20. Mu.l.
In some embodiments, the amplification mix 1 comprises a DNA polymerase, a buffer, magnesium ions, and dntps.
In some embodiments, the ratio of the upstream and downstream primers is 1: 1-2: 1. in some embodiments, the ratio of the upstream and downstream primers is 1:1.
In the amplification system, the concentration of each component is the working concentration.
The procedure for the first round of PCR amplification was: pre-denaturing at 95 ℃ for 10min, denaturing at 98 ℃ for 15s, annealing/extending at 60 ℃ for 5min, and preserving at 10 ℃ infinity; denaturation, annealing/extension was performed for 10 cycles.
In the step 2 of the invention, the magnetic beads are adopted to purify the first PCR amplification product and the second PCR amplification product, and then TE buffer is used for eluting.
In some embodiments, the digestion system comprises:
2. Mu.l of digestion buffer, 1. Mu.l of digestion reaction, 10. Mu.l of purified product after the first round of PCR amplification, and 7. Mu.l of nuclease-free water.
In the present invention, the digestion buffer and the digestion reaction liquid are common in the art, and the digestion buffer and the digestion reaction liquid adopted in the specific examples are Paragon Genomics patent products.
In some embodiments, the amplification system of the second round of PCR amplification comprises:
8 μl of amplification mix 2, 20.4 μl of nuclease-free water, 10 μl of digested and purified reaction product, 400-1000 nM Index primer mix;
the Index primer mixture comprises an Index upstream primer and an Index downstream primer, and the ratio of the Index upstream primer to the Index downstream primer is 1:1. the primer is SEQ ID NO:4 to 5, each primer sequence is as follows:
index upstream primer:
CAAGCAGAAGACGGCATACGAGATCTTCCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T(SEQ ID No.4)
note that: the underlined base is a barcode sequence, which can be replaced; * The base T representing the 3' -end is modified by phosphorylation.
Index downstream primer:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T(SEQ ID No.5)。
note that: * The base T representing the 3' -end is modified by phosphorylation.
The amplification mixture 2 comprises DNA polymerase, buffer solution, magnesium ions and dNTPs.
The procedure for the second round of PCR amplification was: pre-denaturing at 95℃for 10min, denaturing at 98℃for 15s, annealing/extending at 60℃for 75s, preserving at 10℃infinity; denaturation, annealing/extension was performed for 10 cycles.
The amplification mixed solutions 1 and 2 are PCR amplification reagents, including DNA polymerase, buffer solution, magnesium ions and dNTPs. The specific source of the amplification mixed solution is not limited in the invention, and in the specific embodiment, the amplification mixed solutions 1 and 2 are Paragon Genomics patent products.
In the amplification system of the second round of PCR amplification of the present invention, the concentration of the Index primer mixture is 400 to 1000nM, and specifically may be 400nM, 700nM or 1000nM.
The concentration of each component in the amplification system is the working concentration.
In the invention, the index primer adopted in the second PCR amplification is identical to the primer pool panel and SEQ ID NO in the first PCR: 2-3 to form a complete adapter primer.
The invention also provides an RNA library obtained by the construction method.
Compared with the conventional method, the method has the following advantages:
1. the invention can reduce the formation of primer dimer and nonspecific amplification product of RNA amplicon library, so that the library can be more accurately quantified;
2. the library output of the RNA amplicon can be improved, the requirement that the low-quality FFPE sample reaches the minimum denaturation concentration of an illuminea sequencing platform can be met, and the low-quality sample in clinic can enter sequencing and detection analysis; is suitable for popularization and application.
Drawings
FIG. 1 shows a comparison of the method of the present invention with library 2100 data from a conventional method.
Detailed Description
The invention provides an FFPE sample RNA library and a construction method thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
(1) 3 cases of FFPE RNA are extracted and quantified, the sample numbers are (R001, R002 and R003) respectively, and the extraction kit is FFPE DNA/RNA co-extraction kit produced by Wuxi Zhen Yue biotechnology Co-Ltd;
(2) Preparation of spikey plasmid: diluting the plasmid to 4.5X10-5 copies/. Mu.l with ph 8.0TE Buffer for use;
(3) Preparing a primer: the primers on the upstream and downstream of the spikey plasmid are diluted into spikey plasmid primer mother liquor with the final concentration of 50nM according to a certain proportion, and the proportion is 1:1, a step of;
(4) Pretreatment of RNA samples: the reaction system was prepared in 200. Mu.l PCR tubes: 5 μl of the reverse transcription mixture was used, 50ng of each RNA sample was filled to 10 μl with sterilized deionized water, and the reaction tube was reacted at 65deg.C for 5min.
(5) Reverse transcription reaction and purification: the reaction system was prepared in 200. Mu.l PCR tubes: 2. Mu.l of reverse transcriptase mix, 8. Mu.l of reverse transcription buffer 2, 10. Mu.l of pre-treated reaction product; the reaction tube is put into a PCR instrument, and a reverse transcription program is run: 25℃for 10min,42℃for 15min and 70℃for 15min. The purification was carried out using beckmannxp beads 2.2×, and after purification was eluted with 10 μl TE buffer.
(6) And (3) mPCR reaction and purification: the reaction system was assembled in 200. Mu.l PCR tube: 1 μl of spikey plasmid solution, 4 μl of amplification mix 1,5nM primer pool panel,5nM spikey plasmid primer stock, 10 μl of cDNA product, 3 μl nuclease-free water; the reaction tube is put into a PCR instrument, and a mPCR program is run: pre-denaturing at 95 ℃ for 10min, denaturing at 98 ℃ for 15s, annealing/extending at 60 ℃ for 5min, and preserving at 10 ℃ infinity; denaturation, annealing/extension was performed for 10 cycles. The purification was performed using 1.3 Xbeckman Xp beads, and after purification, 10. Mu.l TEbuffer was used.
(7) Digestion reaction and purification: the reaction system was assembled in 200. Mu.l PCR tube: 2. Mu.l of digestion buffer, 1. Mu.l of digestion reaction; 10. Mu.l of the PCR-finished product, 7. Mu.l of nuclease-free water; the reaction tube was put into a PCR instrument and precisely incubated at 37℃for 10min. The purification was performed using 1.3 Xbeckman Xp beads, and after purification, 10. Mu.l TEbuffer was used.
(8)2 nd PCR reaction and purification: the reaction system was assembled in 200. Mu.l PCR tube: 8 μl of amplification mix 2, 20.4 μl of nuclease free water, 10 μl of digested and purified reaction products, 400nM Index primer mix; putting the reaction tube into a PCR instrument, running a PCR program to pre-denature for 10min at 95 ℃, denaturating for 15s at 98 ℃, annealing/extending for 75s, and preserving for 10 ℃ infinity; denaturation, annealing/extension was performed for 10 cycles. The purification was performed using 1 XbeckmannXP beads and eluted with 20. Mu.l TE buffer.
(9) Library quantification and quality inspection: detecting the concentration of the library by using a Qubit instrument; library fragment size detection was performed on the library with agilent tapestation 2100; library quality control data of library construction of the method (library numbers RLIB 1-RLIB 3) of the invention are compared with library quality control data of library construction of the conventional method (note: the conventional method is that spikey plasmids and spikey plasmid primers are not added, the rest of the conventional method is consistent with the method of the invention, library numbers RLIB 4-RLIB 6) are shown in table 1, and a peak diagram of library of the method of the invention is shown in figure 1 with a peak diagram of library of the conventional method 2100.
TABLE 1 comparison of library quality control data for the inventive method with conventional methods
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Wuhan Zhen and medical laboratory Co., ltd
<120> FFPE sample RNA library and construction method thereof
<130> MP21021037
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 157
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgtgaactgt catcggtccg atcaattagt ctagtgtgcg ttattcagat cgagtgagta 60
catgattcgt cagtgtggat caattacagt taggccgctg acacattagt aacgtcggca 120
agcacttagt cgtgtcgtaa gccagtgtgt cgtgtct 157
<210> 2
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cctacacgac gctcttccga tcttgtgaac tgtcatcggt ccg 43
<210> 3
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ttcagacgtg tgctcttccg aaagacacga cacactggct t 41
<210> 4
<211> 66
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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caagcagaag acggcatacg agatcttcct tcgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
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<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58

Claims (7)

1. The construction method of the FFPE sample RNA library is characterized by comprising the following steps of:
step 1: extracting RNA of the FFPE sample, and obtaining cDNA product through reverse transcription;
step 2: adding to the amplification system of the cDNA product a nucleic acid sequence comprising SEQ ID NO:1 and a plasmid for amplifying the DNA fragment shown in SEQ ID NO:1, a primer of the DNA fragment shown in the specification; constructing a library according to a construction method of the RNA sequencing library;
in step 2, the method for constructing the RNA sequencing library comprises the following steps: for said addition of SEQ ID NO:1, performing a first round of PCR amplification by using the DNA fragment amplification system, purifying and digesting, recovering a digested product, performing a second round of PCR amplification, and purifying to obtain an FFPE sample RNA library;
the index primer adopted in the second PCR amplification is identical to the primer pool panel and SEQ ID NO in the first PCR: 2-3 to form a complete joint primer.
2. The method according to claim 1, wherein in step 1, the method further comprises a step of pretreating RNA before the reverse transcription; the pretreatment is as follows: mixing the RNA with the reverse transcription mixed solution, and reacting for 5min at 65 ℃ to obtain a pretreatment reaction product.
3. The method of claim 1, wherein in step 2, the sequence comprising SEQ ID NO:1 is a spikey plasmid, and the amplified sequence of SEQ ID NO:1 comprises the following primers of the DNA fragment: SEQ ID NO:2 and the sequence of SEQ ID NO:3, and a downstream primer shown in 3.
4. The method of claim 1, wherein in step 2, the amplification system of the first round of PCR amplification comprises:
1 μl spikey plasmid, 4 μl amplification mixed solution 1,5 nM-10 nM primer pool for detecting RNA fusion mutation, 5nM~10nM SEQ ID NO: 2-3, namely, 10-30 mu l of cDNA products and supplementing nuclease-free water to 20 mu l;
the amplification mixed solution 1 comprises DNA polymerase, buffer solution, magnesium ions and dNTPs; the ratio of the upstream primer to the downstream primer is 1: 1-2: 1, a step of;
the amplification procedure was: pre-denaturing at 95 ℃ for 10min, denaturing at 98 ℃ for 15s, annealing/extending at 60 ℃ for 5min, and preserving at 10 ℃ infinity; denaturation, annealing/extension was performed for 10 cycles.
5. The method of claim 1, wherein in step 2, the purification is: magnetic bead purification and TE buffer elution are adopted.
6. The method of claim 1, wherein the digestive system comprises:
digestion buffer solution 2 mu l, digestion reaction solution 1 mu l, purified product 10 mu l after first round PCR amplification, and no nuclease water 7 mu l.
7. The construction method according to any one of claims 1 to 6, wherein the amplification system of the second round of PCR amplification comprises:
8 μl of amplification mixed solution 2, 20.4 μl of nuclease-free water, 10 μl of digested and purified reaction product, and 400-1000 nM Index primer mixed solution;
the amplification mixed solution 2 comprises DNA polymerase, buffer solution, magnesium ions and dNTPs;
the amplification procedure was: pre-denaturing at 95 ℃ for 10min, denaturing at 98 ℃ for 15s, annealing/extending at 60 ℃ for 75s, and preserving at 10 ℃ infinity; denaturation, annealing/extension to 10 cycles;
the concentration of the Index primer mixture is 400-1000 nM.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107385018A (en) * 2017-06-22 2017-11-24 哈尔滨博泰生物科技有限公司 A kind of method and its application of the RNA high-throughput sequencing libraries structure of optimization
CN109505012A (en) * 2019-01-15 2019-03-22 依科赛生物科技（太仓）有限公司 A kind of kit of the mRNA bis- generations sequencing library building for FFPE sample and its application
CN110656156A (en) * 2019-10-14 2020-01-07 湖南大地同年生物科技有限公司 Ultralow frequency mutation nucleic acid fragment detection method, library construction method, primer design method and reagent
CN110951827A (en) * 2018-12-28 2020-04-03 广州表观生物科技有限公司 Rapid construction method and application of transcriptome sequencing library
CN111549380A (en) * 2020-05-22 2020-08-18 南京诺唯赞生物科技股份有限公司 Kit for constructing double-stranded RNA sequencing library and application thereof
CN111808854A (en) * 2020-07-09 2020-10-23 中国农业科学院农业基因组研究所 Balanced joint with molecular bar code and method for quickly constructing transcriptome library
CN113265374A (en) * 2021-05-20 2021-08-17 云南贺尔思细胞生物技术有限公司 Preparation method of hair follicle stem cells

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107385018A (en) * 2017-06-22 2017-11-24 哈尔滨博泰生物科技有限公司 A kind of method and its application of the RNA high-throughput sequencing libraries structure of optimization
CN110951827A (en) * 2018-12-28 2020-04-03 广州表观生物科技有限公司 Rapid construction method and application of transcriptome sequencing library
CN109505012A (en) * 2019-01-15 2019-03-22 依科赛生物科技（太仓）有限公司 A kind of kit of the mRNA bis- generations sequencing library building for FFPE sample and its application
CN110656156A (en) * 2019-10-14 2020-01-07 湖南大地同年生物科技有限公司 Ultralow frequency mutation nucleic acid fragment detection method, library construction method, primer design method and reagent
CN111549380A (en) * 2020-05-22 2020-08-18 南京诺唯赞生物科技股份有限公司 Kit for constructing double-stranded RNA sequencing library and application thereof
CN111808854A (en) * 2020-07-09 2020-10-23 中国农业科学院农业基因组研究所 Balanced joint with molecular bar code and method for quickly constructing transcriptome library
CN113265374A (en) * 2021-05-20 2021-08-17 云南贺尔思细胞生物技术有限公司 Preparation method of hair follicle stem cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Synthetic spike-in standards for RNA-seq experiments;Jiang等;Genome Res.;第21卷(第9期);1543–1551 *
登录号：KC702168.1;GenBank;GenBank;604-760bp *

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