CN114134206A - FFPE sample RNA library and construction method thereof - Google Patents

FFPE sample RNA library and construction method thereof Download PDF

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CN114134206A
CN114134206A CN202111476559.8A CN202111476559A CN114134206A CN 114134206 A CN114134206 A CN 114134206A CN 202111476559 A CN202111476559 A CN 202111476559A CN 114134206 A CN114134206 A CN 114134206A
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primer
amplification
library
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rna
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CN114134206B (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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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 in the process of constructing the pool, and the artificial sequence and the primer pool panel for detecting RNA fusion mutation are effectively amplified and constructed together, so that the ex-pool amount is ensured, meanwhile, due to the dominant amplification of the plasmid, the probability of nonspecific collision, combination and amplification of the panel primer is reduced, and the generation of primer and joint dimer is reduced.

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, but samples (FFPE) which are Fixed by Formalin and Embedded by paraffin can be kept for a long time at normal temperature, so that the paraffin embedding preservation of the tumor tissues becomes the most common sample storage means in hospitals. The formalin is fixed to easily degrade nucleic acid in tissues to different degrees and crosslink molecules, the degradation of the nucleic acid is further accelerated in the high-temperature infiltration process of paraffin, the storage time and environment have great influence on the nucleic acid in a sample, high-quality nucleic acid is extracted from paraffin sections, and a library which is complete in information and can be used for subsequent RNA research is constructed, so that the problems are quite difficult.
Complexity of rnafpe samples: 1. will crosslink with proteins and other cellular components; 2. oxidation of formalin to formic acid can be caused over time, which causes depurination of bases and breakage of nucleic acid chains, resulting in low extraction and RNA quality; 3. is easily polluted by PCR inhibitor, and the sample contains the PCR inhibitor. The complexity of the method causes the difficulty in constructing the RNA library based on amplicon methodology library construction sequencing, and the library yield is low, so the library construction success rate is low; primer and linker dimers are more severe and the library quality is poor.
The Illumina Miniseq and Miseq sequencing platform has the minimum requirement of 2nM on the denaturation of the upper computer library, the denaturation effect is poor when the denaturation effect is lower than 2nM, and the sequencing success rate is low; it was therefore specified that the library which can be used for the sequencing of the on-board library should have a certain export concentration (for example, the panel to which this patent refers requires an RNA library export concentration of. gtoreq.0.4 ng/. mu.l); in addition, due to the complexity of the RNA sample, excessively high primer dimer and linker dimer are easily introduced in the database construction process of the FFPE RNA sample with low quality. Therefore, in clinical application, the library quality of the conventional amplicon library construction method is low, and the library construction power is low. In order to solve the problems of amplicon library quality (increasing the proportion of target fragments of the library, reducing the proportion of non-main peak fragments such as primer and linker dimer) and low RNA library construction power (low RNA ex-warehouse concentration), technologists propose methods such as increasing the amplification cycle number and increasing the sequencing depth.
The defects and shortcomings of the prior art are as follows:
1. increasing the amplification cycle number can improve the RNA library construction power, but too high primer dimer (dimer) and linker dimer are easily introduced in the low-quality FFPE RNA sample library construction process, and the too high linker dimer can cause inaccurate quantification of the main peak of the library by using a method for quantifying the qubit, and influence the accurate pooling of the library.
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 can not be subjected to sequencing of the illumina platform due to low ex-warehouse concentration.
Disclosure of Invention
In view of the above, the present invention provides a method for constructing an FFPE sample RNA library. In the method, in the library building process, a section of artificial sequence (DNA fragment shown in SEQ ID NO: 1) is added to effectively amplify and build a library together with a primer pool panel which is mixed with plasmid upstream and downstream primers and can be used for detecting RNA fusion mutation, so that the library building success rate and the quality of an RNA amplicon library 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 a cDNA product through reverse transcription;
step 2: adding a nucleic acid sequence containing SEQ ID NO: 1 and the plasmid for amplifying the DNA fragment shown in SEQ ID NO: 1, and primers of the DNA fragments shown in the specification; the library was constructed according to the construction method of RNA sequencing library.
The invention incorporates a segment of DNA fragment artificial sequence in the process of library construction, namely a DNA fragment artificial sequence comprising SEQ ID NO: 1, and a plasmid containing a fragment shown by SEQ ID NO: and (3) effectively amplifying and constructing a library together with a primer pool panel of the upstream and downstream primers which can be used for detecting RNA fusion mutation. The DNA fragment has NO homologous sequence with the human genome, can be artificially synthesized, and can also be synthesized by using a spikein-09 plasmid as a template, wherein the DNA fragment is represented by SEQ ID NO: 2-3, and amplifying. The sequences are as follows:
SEQ ID No.1：
TGTGAACTGTCATCGGTCCGATCAATTAGTCTAGTGTGCGTTATTCAGATCGAGTGAGTACATGATTCGTCAGTGTGGATCAATTACAGTTAGGCCGCTGACACATTAGTAACGTCGGCAAGCACTTAGTCGTGTCGTAAGCCAGTGTGTCGTGTCT。
an upstream primer 1:
5’-CCTACACGACGCTCTTCCGATCTTGTGAACTGTCATCGGTCCG-3’
(SEQ ID No.2)；
a downstream primer 2:
5’-TTCAGACGTGTGCTCTTCCGAAAGACACGACACACTGGCTT-3’(SEQ ID No.3)。
wherein, the underlined sequence at the 5' end of the upstream and downstream primers is tail sequence, and the sequence can be combined with the 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 plasmid spikein-09 with a copy number of 4.5X 10^ 5.
In the step 1, a step of pretreating RNA is further included before the reverse transcription; the pretreatment comprises the following steps: and mixing the RNA and 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 for constructing the RNA sequencing library comprises: adding the sequence shown in SEQ ID NO: 1, performing first round PCR amplification, purifying and digesting, recovering a digestion product, performing second round 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 mul of spikein plasmid, 4 mul of amplification mixed liquor 1, 5 nM-10 nM primer pool for detecting RNA fusion mutation, 5 nM-10 nM SEQ ID NO: 2-3, 10-30 mu l of cDNA product, and adding nuclease-free water to 20 mu l.
In some embodiments, the amplification mixture 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 above amplification system, the concentrations of the components are working concentrations.
The procedure for the first round of PCR amplification was: pre-denaturation at 95 deg.C for 10min, denaturation at 98 deg.C for 15s, annealing/extension at 60 deg.C for 5min, and storing at 10 deg.C for infinity; denaturation, annealing/extension were 10 cycles.
In step 2 of the invention, magnetic beads are used for purifying the first round PCR amplification product and the second round PCR amplification product, and then TE buffer is used for elution.
In some embodiments, the digestion system comprises:
2 ul of digestion buffer, 1 ul of digestion reaction, 10 ul of purified product after the first round of PCR amplification, and 7 ul of nuclease-free water.
In the invention, the digestion buffer solution and the digestion reaction solution are common in the field, and the digestion buffer solution and the digestion reaction solution adopted in the specific embodiment are Paragon Genomics patent products.
In some embodiments, the amplification system for the second round of PCR amplification comprises:
8 mul of amplification mixed solution 2, 20.4 mul of nuclease-free water, 10 mul of digested and purified reaction product and 400-1000 nM Index primer mixed solution;
the Index primer mixed solution comprises an Index upstream primer and an Index downstream primer, wherein the Index upstream primer and the Index downstream primer are in a ratio of 1: 1. the primer is SEQ ID NO: 4-5, and the primer sequences are as follows:
index upstream primer:
CAAGCAGAAGACGGCATACGAGATCTTCCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T(SEQ ID No.4)
note: underlined bases are barcode sequences, which are replaceable; indicates that base T at the 3' end was modified by phosphorylation.
Index downstream primer:
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T(SEQ ID No.5)。
note: indicates that base T at the 3' end was modified by phosphorylation.
The amplification mixture 2 includes DNA polymerase, buffer, magnesium ions and dNTPs.
The procedure for the second round of PCR amplification was: pre-denaturation at 95 ℃ for 10min, denaturation at 98 ℃ for 15s, annealing/extension at 60 ℃ for 75s, and preserving at 10 ℃ for infinity; denaturation, annealing/extension were 10 cycles.
The amplification mixed solutions 1 and 2 are PCR amplification reagents, including DNA polymerase, buffer solution, magnesium ions and dNTP. The invention has no limitation on the specific source of the amplification mixed solution, and in the specific embodiment, the amplification mixed solutions 1 and 2 adopted are Paragon Genomics patent products.
In the amplification system of the second round of PCR amplification, the concentration of the Index primer mixture is 400-1000 nM, and specifically 400nM, 700nM or 1000 nM.
The concentrations of the components in the amplification system are working concentrations.
In the invention, the index primers used in the second round of PCR amplification and the primer pools panel and SEQ ID NO: 2-3 form a complete adaptor 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 non-specific amplification product of the RNA amplicon library, so that the library can be quantified more accurately;
2. the method can improve the output of the RNA amplicon library, meet the requirement that the low-quality FFPE sample reaches the lowest denaturation concentration of an illumina sequencing platform, and enable the low-quality sample in clinic to enter sequencing, detection and analysis; is suitable for popularization and application.
Drawings
FIG. 1 shows a comparison of the data for the library 2100 of the present invention and conventional methods.
Detailed Description
The invention provides an FFPE sample RNA library and a construction method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention 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) Extracting and quantifying 3 cases of FFPE RNA, wherein the sample numbers are (R001, R002 and R003), and the extraction kit adopts an FFPE DNA/RNA co-extraction kit produced by Wuxi Zheyue Biotech limited;
(2) preparation of spikein plasmid: the plasmid is diluted to 4.5 x 10^5 copies/mu l for later use by using ph 8.0TE Buffer;
(3) preparing a primer: diluting the upstream and downstream primers of the spikein plasmid into a spikein plasmid primer mother solution with the final concentration of 50nM according to a certain proportion for later use, wherein the proportion is 1: 1;
(4) RNA sample pretreatment: prepare the reaction system in a 200. mu.l PCR tube: mu.l of the mixture was reverse transcribed, 50ng of each RNA sample was made up to 10. mu.l with sterilized deionized water, and the reaction tube was allowed to react at 65 ℃ for 5 min.
(5) Reverse transcription reaction and purification: prepare the reaction system in a 200. mu.l PCR tube: mu.l of reverse transcriptase mixed liquor, 8 mu.l of reverse transcription buffer solution 2 and 10 mu.l of pretreatment reaction product; placing the reaction tube into a PCR instrument, and operating a reverse transcription program: 10min at 25 ℃, 15min at 42 ℃ and 15min at 70 ℃. Purification was performed using beckmanXp magnetic beads 2.2 ×, and after purification, elution was performed using 10 μ l of TE buffer for use.
(6) mPCR reaction and purification: in a 200. mu.l PCR tube reaction: mu.l of spikein plasmid solution, 4. mu.l of amplification mix 1, 5nM primer pool panel, 5nM spikein plasmid primer stock, 10. mu.l of cDNA product, 3. mu.l of nuclease-free water; the reaction tube is placed in a PCR instrument, and the mPCR program is run: pre-denaturation at 95 deg.C for 10min, denaturation at 98 deg.C for 15s, annealing/extension at 60 deg.C for 5min, and storing at 10 deg.C for infinity; denaturation, annealing/extension was 10 cycles. The purification was performed using 1.3 Xbeckman Xp magnetic beads, and then eluted with 10. mu.l of TEbuffer for further use.
(7) Digestion reaction and purification: in a 200. mu.l PCR tube reaction: mu.l digestion buffer solution and 1 mu.l digestion reaction solution; the product was purified after 10. mu.l mPCR, 7. mu.l nuclease free water; the reaction tube is placed into a PCR instrument and incubated for 10min at 37 ℃. The purification was performed using 1.3 Xbeckman Xp magnetic beads, and then eluted with 10. mu.l of TEbuffer for further use.
(8)2ndPCR reaction and purification: in a 200. mu.l PCR tube reaction: mu.l amplification mixture 2, 20.4. mu.l nuclease-free water, 10. mu.l digestion-purified reaction product, 400nM Index primer mixture; placing the reaction tube into a PCR instrument, running a PCR program to perform pre-denaturation at 95 ℃ for 10min, denaturation at 98 ℃ for 15s, annealing/extension for 75s, and storing at 10 ℃ for infinity; denaturation, annealing/extension was 10 cycles. The purification was performed using 1 XbeckmanXp magnetic beads, and the purification was performed using 20. mu.l of TE buffer.
(9) Library quantification and quality control: carrying out concentration detection on the library by using a Qubit instrument; library fragment size detection of the library with agilent tapestation 2100; a comparison of the library quality control data of the library constructed by the method of the present invention (library numbers RLIB 1-RLIB 3) and the conventional method (note: the conventional method is not added with spikein plasmid and spikein plasmid primers, and the rest is consistent with the method of the present invention, library numbers RLIB 4-RLIB 6) is shown in Table 1, and the library peak diagram of the method of the present invention and the conventional method 2100 is shown in FIG. 1.
TABLE 1 comparison of library quality control data between the methods of the invention and conventional methods
Figure BDA0003393734660000061
Figure BDA0003393734660000071
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
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<120> FFPE sample RNA library and construction method thereof
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Claims (10)

1. A construction method of an FFPE sample RNA library is characterized by comprising the following steps:
step 1: extracting RNA of the FFPE sample, and obtaining a cDNA product through reverse transcription;
step 2: adding a nucleic acid sequence containing SEQ ID NO: 1 and the plasmid for amplifying the DNA fragment shown in SEQ ID NO: 1, and primers of the DNA fragments shown in the specification; the library was constructed according to the construction method of RNA sequencing library.
2. The method according to claim 1, wherein step 1 further comprises a step of pretreating the RNA before the reverse transcription; the pretreatment comprises the following steps: and mixing the RNA and the reverse transcription mixed solution, and reacting for 5min at 65 ℃ to obtain a pretreatment reaction product.
3. The method of claim 1, wherein the step 2, the method of constructing the RNA sequencing library comprises: adding the sequence shown in SEQ ID NO: 1, performing first round PCR amplification, purifying and digesting, recovering a digestion product, performing second round PCR amplification, and purifying to obtain an FFPE sample RNA library.
4. The method of claim 1, wherein in step 2, the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 is a spikein plasmid, and the amplified DNA fragment shown in SEQ ID NO: 1 comprises the following components: SEQ ID NO: 2 and the upstream primer shown in SEQ ID NO: 3, or a reverse primer as shown in the figure.
5. The construction method according to any one of claims 1 to 4, wherein in the step 2, the amplification system of the first round of PCR amplification comprises:
1 mul of spikein plasmid, 4 mul of amplification mixed liquor 1, 5 nM-10 nM primer pool for detecting RNAfusion mutation, 5 nM-10 nM SEQ ID NO: 2-3, 10-30 mu l of cDNA product, and supplementing nuclease-free water to 20 mu l;
the amplification mixed solution 1 comprises DNA polymerase, buffer solution, magnesium ions and dNTP; the ratio of the upstream primer to the downstream primer is 1: 1-2: 1;
the amplification procedure was: pre-denaturation at 95 deg.C for 10min, denaturation at 98 deg.C for 15s, annealing/extension at 60 deg.C for 5min, and storing at 10 deg.C for infinity; denaturation, annealing/extension were 10 cycles.
6. The construction method according to claim 3, wherein in step 2, the purification is: magnetic beads are used for purification, and TEbuffer is used for elution.
7. The construction method according to claim 3, wherein the digestion system comprises:
2 ul of digestion buffer, 1 ul of digestion reaction, 10 ul of purified product after the first round of PCR amplification, and 7 ul of nuclease-free water.
8. The construction method according to any one of claims 3 to 8, wherein the amplification system for the second round of PCR amplification comprises:
8 mul of amplification mixed solution 2, 20.4 mul of nuclease-free water, 10 mul 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 dNTP;
the amplification procedure was: pre-denaturation at 95 ℃ for 10min, denaturation at 98 ℃ for 15s, annealing/extension at 60 ℃ for 75s, and preservation at 10 ℃ for infinity; denaturation, annealing/extension for 10 cycles;
the concentration of the Index primer mixture is 400-1000 nM.
9. The construction method according to any one of claims 1 to 8, wherein index primers used in the second round of PCR amplification are identical to the primer pools panel and SEQ ID NO: 2-3 form a complete adaptor primer.
10. An RNA library obtained by the construction method according to any one of claims 1 to 9.
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JIANG等: "Synthetic spike-in standards for RNA-seq experiments", GENOME RES., vol. 21, no. 9, pages 1543, XP055152443, DOI: 10.1101/gr.121095.111 *

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