CN112063689A

CN112063689A - Efficient RNA reverse transcription method and RNA and DNA synchronous library building method

Info

Publication number: CN112063689A
Application number: CN202010849568.6A
Authority: CN
Inventors: 杜玉龙; 李夏静; 陈才夫
Original assignee: Shanghai 3D Medicines Co Ltd
Current assignee: Shanghai 3D Medicines Co Ltd
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-12-11

Abstract

The invention relates to a high-efficiency RNA reverse transcription method and a synchronous RNA and DNA library construction method, wherein the reverse transcription is carried out after DNA and RNA are mixed, the RNA reverse transcription efficiency is improved, all reverse transcription products are directly and synchronously constructed, and an rhPCR amplification method is adopted in the library construction process, so that the possibility of dimer generation is reduced, the risk of secondary library construction is effectively avoided, and the operation is simpler.

Description

Efficient RNA reverse transcription method and RNA and DNA synchronous library building method

Technical Field

The invention relates to the technical field of gene sequencing, in particular to a high-efficiency RNA reverse transcription method and a RNA and DNA synchronous library building method.

Background

Next Generation Sequencing (NGS) technology has revolutionized the field of genomics over the past decade. Each NGS run typically generates thousands of megabits of sequence information on hundreds of thousands to billions of DNA templates in parallel for a single sequencing run. The cost for human genome sequencing has currently reached a benchmark of $ 1000. The low cost and high throughput of NGS technology enables one to use nucleic acid sequencing as a clinical tool.

The genetic traits of an organism are determined primarily by nucleic acids, including DNA and RNA. In recent years, with the development of molecular biology and various omics technologies, comparative research on nucleic acid sequences has gradually become a basic means for disease diagnosis, genetic variation, regulation and control mechanism, and disease prevalence rule analysis. At present, only one of DNA or RNA is selected for detection in a sample for gene detection, and the two have large differences in structure and properties, so that the flow from extraction to detection can be distinguished. For the detection of the information required, in particular of the mutations, both DNA and RNA will generally be present, so that the detection of only one of them will result in the omission of valuable information from the other, but in terms of technical means, the detection of a single nucleic acid type is indeed easier to implement and to optimize. In practical assays, separate detection of DNA and RNA means greater sample requirements. Meanwhile, some mutation or fusion detection with low content is involved in early disease screening, which may mean increase of detection difficulty or instability of detection. The simultaneous detection of DNA and RNA in the same system provides a feasible solution to the above-mentioned problems.

Amplicon Sequencing (amplification Sequencing) is a Sequencing method that only performs Sequencing studies on the target region. The method comprises the steps of designing a primer of a genome region of interest, carrying out PCR amplification, enriching a target region, then carrying out library construction aiming at a PCR product with a specific length or a captured fragment, carrying out high-throughput sequencing, and analyzing variation in a sequence. In the traditional NGS library construction of DNA or RNA, the DNA and RNA are separately constructed, and the method has the advantages of single detection project, high material consumption, high labor and high reagent cost. At present, the main method for detecting SNV sites and Fusion sites is to separately build libraries, because SNV detection is based on DNA, and Fusion detection is based on RNA, the former is to directly build a DNA library, and the latter needs to be firstly reversely transcribed into cDNA and then build a cDNA library, the two need to be separately operated, and then simultaneously, the operation is complicated because the SNV detection and the Fusion detection are simultaneously operated on a computer for sequencing. Because two libraries need to be constructed, the library construction process is complicated, and the risk of library construction failure is relatively increased. Meanwhile, the database construction technology for sequencing the amplicon generally uses a large amount of primers, so that a large amount of primer dimers are easily generated, and the complexity of operation and the required amount of detection samples are increased.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a high-efficiency RNA reverse transcription method and a RNA and DNA synchronous library construction method, the RNA reverse transcription efficiency is improved by mixing DNA and RNA and then performing reverse transcription together, all reverse transcription products are directly and synchronously constructed, and an rhPCR amplification method is adopted in the library construction process, so that the possibility of dimer generation is reduced, the risk of secondary library construction is effectively avoided, and the operation is simpler.

In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:

a method for reverse transcription of RNA with high efficiency, comprising:

and carrying out reverse transcription operation on a mixed sample formed by quantitatively mixing the RNA sample and the DNA sample to obtain a mixed reverse transcription product.

Further, the quantitatively mixing the RNA sample and the DNA sample comprises mixing the RNA and the DNA in a mass ratio of 1: 1.

The invention also relates to a high-efficiency RNA reverse transcription system, which is characterized by comprising DNA and RNA at least simultaneously.

The invention also relates to a method for synchronously constructing the library of RNA and DNA, which is characterized by comprising the following steps:

carrying out reverse transcription on a mixed sample formed by quantitatively mixing an RNA sample and a DNA sample to obtain a mixed reverse transcription product;

and applying the mixed reverse transcription product to a subsequent library construction process.

Further, the reverse transcription procedure involves the use of the same reverse transcription reagents as for reverse transcription of the RNA sample alone.

Further, the subsequent library construction process comprises targeted region amplification, first purification, linker primer amplification and second purification.

Further, the targeted region amplification comprises adding the mixed reverse transcription product into a rhAmpSeq reaction system to perform a PCR reaction; the rhAmpSeq reaction system comprises rhPrimer primers, RNase H2 enzyme and PCR reaction reagents.

The invention also relates to a Fusion mutation detection method, which is characterized by comprising the following steps:

constructing a library required for detection by using the RNA and DNA synchronous library construction method as described above;

and sequencing the library and analyzing biological information to obtain Fusion mutation information.

The invention also relates to a reverse transcription kit, which is characterized by comprising the reverse transcription system.

The invention also relates to a kit for synchronously constructing the RNA and DNA library, which is characterized by at least comprising a mixed sample formed by quantitatively mixing the RNA sample and the DNA sample, rhPrimer and RNase H2 enzyme. The synchronous kit can also comprise other conventional PCR reaction reagents and library building reagents.

The invention has the beneficial effects that:

by adopting the efficient RNA reverse transcription method and the RNA and DNA synchronous library building method, through the special operation of adding the DNA sample in the RNA reverse transcription operation stage, the experiment verifies that compared with the traditional single RNA sample operation, the RNA reverse transcription efficiency can be obviously improved, so that the generation of primer dimers in the subsequent steps can be reduced, the library building operation process can be simplified, and the DNA and RNA synchronous library building can be realized; the rhPrimer is matched with RNase H2 enzyme to block extension of primer dimer, reduce amplification of primer dimer and improve amplification quality of library.

Detailed Description

For a clearer understanding of the contents of the present invention, reference will be made to the following examples.

The current amplicon sequencing technology usually uses a large amount of primers, which easily causes the generation of a large amount of primer dimers, and the common solution is to split the reaction system from one tube to multiple tubes, thereby reducing the number of primers in each tube of reaction and reducing the generation of primer dimers. However, this solution would significantly increase the complexity of the library construction operation, and the requirement for sample demand is high.

According to the invention, DNA is innovatively and directly added into an RNA reverse transcription system, so that the RNA reverse transcription efficiency can be obviously improved, the library building operation steps can be simplified, and synchronous library building and detection of DNA and RNA are realized; the method of rhAmpSeq PCR (Integrated DNA Technologies, rhPCR method of IDT company, https:///sfvideo. blob. core. windows. net/morphology/docs/default-source/protocol/rhpc-protocol. pdf.

The rhAmpSeq reaction system of the present invention includes rhPrimer primers, i.e., blocked-cleavable rhPCR primers (RNase H-Dependent PCR, see U.S. patent application No. US12433896, publication No. US20090325169a 1).

The process of the preferred embodiment of the method for synchronously constructing the library of RNA and DNA mainly comprises the following steps:

1) obtaining DNA samples and RNA samples, for example, preferably a method of extracting a paraffin-embedded sample (FFPE) or a Qiazol Lysis Reagent (cat # by Qiagen) with a DNA and RNA extraction kit (cat # 80234) by Qiagen is used for the preparation of a DNA sample and an RNA sample, and the method is preferably performed by subjecting a paraffin-embedded sample (FFPE) or a Qiazol Lysis Reagent (cat # by Qiagen: 5346994) was extracted from the cell line RNA.

2) The DNA and RNA samples obtained are quantified, for example, preferably using the Qubit dsDNA HS Assay Kit (Invitrogen, cat # Q32854) and the Qubit RNA HS Assay Kit (Invitrogen, cat # Q32855), using the instructions of Qubit 2.0, ThermoFisher corporation.

3) The mixed sample obtained by quantitatively mixing the RNA sample and the DNA sample is subjected to a reverse transcription operation to obtain a mixed reverse transcription product, and preferably a reverse transcription kit (cat no: RK20420) is subjected to reverse transcription operation in a 10-L reaction system, and the specific reaction system is preferably shown in Table 1.

TABLE 1 reverse transcription reaction System

The volume values X and Y of the RNA sample and the DNA sample can be determined according to the detection requirement, for example, preferably, X and Y are equal to each other and are 1 muL, and at the moment, the deionized water is 5 muL. The reaction program of the reverse transcription follows the conventional operation steps, for example, 25 ℃ and 5min can be selected; 30min at 42 ℃; 5min at 80 ℃; the program of hold was set to perform the reverse transcription reaction at 4 ℃.

4) And carrying out target region amplification on the mixed reverse transcription product. Preferably, 10. mu.L of the mixed reverse transcription product obtained in the above step is added into a 20. mu.L rhAmpSeq reaction system, and a Fusion primer or a detection SNV-Indel primer is added at the same time to carry out PCR1 reaction, wherein the specific reaction system is shown in Table 2 (wherein, 4X rhAmpSeq Library Mix 1, SNV-Indel & Fusion _ FWD primers and SNV-Indel & Fusion _ REV primers are rhPrimer primers from IDT company).

TABLE 2 target region amplification reaction System

A preferred reaction procedure may be as shown in table 3.

TABLE 3 target region amplification reaction procedure

The target amplification product obtained by the above reaction procedure needs to be purified. Preferably, the purification of the target product can be carried out by using Agencour AMPure XP beads and 80% ethanol (now prepared), and the specific steps comprise:

a. adding 30 mu L of magnetic beads (1.5x magnetic beads) into the PCR tube filled with the reaction solution, shaking and uniformly mixing, centrifuging for a short time, and incubating at room temperature for 10 min;

b. placing on a magnetic frame, standing until the liquid is clear, and removing the supernatant by using a pipette (taking care not to touch magnetic beads and keeping the PCR tube on the magnetic frame all the time);

c. adding 200 mu L of 80% ethanol into the PCR tube, standing for 30s, and removing the supernatant;

d. repeating c;

e. sucking liquid in the PCR tube as clean as possible by using a 10-mu-L gun head, and standing at room temperature until the magnetic beads are dried;

f. adding 22 mu L of nuclease-free water, taking down the PCR tube from the magnetic frame, shaking and mixing uniformly, and centrifuging for a short time;

g. incubating at room temperature for 5min, placing the PCR tube on a magnetic frame, standing until the liquid is clear, sucking 20 μ L of supernatant, and adding into a new PCR tube;

h. adding 30 mu L of magnetic beads into the PCR tube filled with the supernatant, uniformly mixing by shaking, carrying out short-time centrifugation, and then incubating at room temperature for 10 min;

i. standing on a magnetic frame until the liquid is clear, and removing the supernatant by using a pipette (taking care not to touch the magnetic beads and keeping the PCR tube on the magnetic frame all the time);

g. adding 200 mu L of 80% ethanol into the PCR tube, standing for 30s, and removing the supernatant;

k. repeating g;

l, sucking liquid in the PCR tube as clean as possible by using a 10-mu-L gun head, and standing at room temperature until the magnetic beads are dried;

m. adding 20 μ L of reaction liquid prepared according to the components shown in Table 4 into the dried magnetic beads, shaking and uniformly mixing, and incubating at room temperature for 3 min;

TABLE 4 reaction liquid composition in purification of target amplification product

And n, placing the mixture on a magnetic frame, standing until the liquid is clear, transferring the supernatant into a new PCR tube, and then carrying out amplification according to a reaction procedure shown in the table 5 to obtain a purified product.

TABLE 5 reaction procedure for purification of target amplification product

5) And (5) purifying the target library. The Agencour AMPure XP beads are selected to purify a target product library by 80% ethanol (used for preparation), and the purified library can be subjected to quantitative quality inspection. The specific purification steps include:

a. adding 20 mu L of magnetic beads (1.0x magnetic beads) into the PCR tube filled with the reaction solution, shaking and uniformly mixing, centrifuging for a short time, and incubating at room temperature for 10 min;

d. repeating c;

g. and (3) incubating at room temperature for 5min, placing the PCR tube on a magnetic frame, standing until the liquid is clear, sucking 20 mu L of supernatant, and adding the supernatant into a new 1.5ml EP tube to obtain the library.

h. The constructed library was subjected to Qubit quantification and lab chip fragment analysis.

6) And performing on-machine sequencing on the obtained library, and performing biological process analysis.

By adopting the steps, the synchronous library building and analysis of the DNA and the RNA can be realized, compared with the method that the RNA is firstly reversely transcribed and then added with the DNA, the reverse transcription efficiency of the RNA is improved, and simultaneously, the flow is simplified, so that the SNV-indel and Fusion co-detection becomes possible; using the rhPCR method, RNase H2 only works when rhPrimer binds to the correct DNA template, cleaves RNA bases, and then extends. When the two primers bind, RNase H2 does not function, thereby making primer dimers non-extended and reducing amplification of primer dimers.

To further illustrate the better technical effect of the simultaneous library-building method of RNA and DNA of the present invention compared to the prior art, a specific comparative example is used for comparison.

1) Sample selection: 7 samples of Fusion positive cell lines, 2 samples of Fusion positive FFPE and 1 sample of SNV negative were selected, and the extracted RNA and DNA samples were diluted to 50 ng/. mu.L, and the details are shown in Table 6.

Table 6 example sample list

2) Reverse transcription of RNA and DNA: set up 2 sets of experiments, first set: RNA of 9 Fusion positive samples and negative DNA samples were subjected to reverse transcription, 10. mu.L of the reaction system was used, 9 reactions were performed in total, and the reverse transcription operation was carried out using a reverse transcription kit (cat. RK20420) provided by ABClonal, and the reaction system is shown in Table 7.

TABLE 7 example first set of reverse transcription reaction System

A second set of 9 Fusion positive samples were subjected to reverse transcription of RNA alone in a 10. mu.L reaction system, which was shown in Table 8, for a total of 9 reactions, using a reverse transcription kit (cat. RK20420) provided by ABClonal.

TABLE 8 example second set of reverse transcription reaction System

In total, 18 reverse transcription reactions were performed, and the reverse transcription PCR step was performed according to the reaction procedure (25 ℃, 5 min; 42 ℃, 30 min; 80 ℃, 5 min; 4 ℃, hold).

3) Amplification of the target region: adding 10 mu L of the reverse transcription reaction product of the first group into a 20 mu L reaction system of the rhAmpSeq, and adding a Fusion primer or simultaneously adding an SNV-Indel primer to carry out PCR1 reaction; the reaction system is shown in Table 9.

TABLE 9 example first set of target region amplification reaction System

Adding 10 mu L of reverse transcription reaction product into a 20 mu L rhAmpSeq reaction system, and simultaneously adding SNV-Indel and Fusion primer to carry out PCR1 reaction; the reaction system is shown in Table 10.

TABLE 10 example second set of target region amplification reaction systems

A total of 18 reactions were performed, and PCR1 target region amplification was performed according to the reaction procedure shown in Table 11.

TABLE 11 example target region amplification reaction procedure

The primers used to detect Fusion mutations are shown in the table below. All primers used were blocking-cleavable rhPCR primers (from Integrated DNA Technologies, IDT) with rA, rG, rC, rU in the sequence each representing one RNA base, 3SpC3 attached to the 3 'end of each sequence as a means for 3' end nucleic acid blocking (Blocker).

TABLE 12 primer sequences used in the examples

4) And (3) purifying a target amplification product: the Agencour AMPure XP beads are selected and used for purifying a target product by using 80% ethanol (which is currently used for preparation).

The specific purification steps are as follows:

d. repeating c;

k. repeating g;

5) Purification of the target library: the method comprises the steps of selecting Agencour AMPure XP beads, purifying a target product by using 80% ethanol (used for preparation), and carrying out quantitative quality inspection on a purified library.

The specific purification steps are as follows:

d. repeating c;

6) Nova on-machine sequencing was performed on the library and a biogenetic process analysis was performed.

7) The experimental results are as follows:

a. the library quantification results are shown in table 13:

TABLE 13 example library quantification results

Table 13 results show the comparison of the co-reverse transcription of DNA and RNA (co-RT, present invention) with the split reverse transcription (split RT, prior art), with identical primers for both groups, SNV-Indel & Fuison _ FWD (100nM) and SNV-Indel & Fusion _ REV (100 nM). Wherein the column <200bp (write) represents the percentage of all remaining primers and primer dimers in the library over the entire library, consisting mainly of primer dimers (most of which have been removed during purification because no dimer was formed), the library concentration for separate transcription of DNA and RNA is generally lower than for co-transcription of RNA and DNA, and the proportion of primer dimers is generally higher than for co-transcription of DNA and RNA, in terms of library concentration and proportion of primer dimers.

The results of comparison of the number of fusion positive reads are shown in Table 14.

TABLE 14 comparison of the number of Fusion positive reads detected in the examples

From the sequencing results, Fusion positive fusions in which DNA and RNA were separately transcribed (prior art) generally detected lower numbers of reads than when DNA and RNA were co-reverse transcribed (present invention).

In conclusion, when a Fusion form is detected, DNA is added into a reverse transcription system, so that the RNA reverse transcription efficiency can be effectively improved, and the accuracy of Fusion detection on the RNA level is improved.

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 changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

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Claims

1. A method for reverse transcription of RNA with high efficiency, comprising:

2. The method of claim 1, wherein quantitatively mixing the RNA sample with the DNA sample comprises mixing RNA and DNA in a mass ratio of 1: 1.

3. A high efficiency RNA reverse transcription system, wherein the reverse transcription system comprises at least DNA and RNA simultaneously.

4. A method for simultaneous construction of a library of RNA and DNA, comprising:

5. The method of claim 4, wherein quantitatively mixing the RNA sample with the DNA sample comprises mixing the RNA and the DNA in a mass ratio of 1: 1.

6. The method of claim 4, wherein the reverse transcription comprises using the same reverse transcription reagents as reverse transcription of the RNA sample alone.

7. The method of claim 4, wherein the subsequent library construction process comprises targeted region amplification, first purification, adapter primer amplification, and second purification.

8. The method of claim 7, wherein the targeted region amplification comprises adding the mixed reverse transcription product to a rhAmpSeq reaction system for a PCR reaction; the rhAmpSeq reaction system comprises rhPrimer primers, RNase H2 enzyme and PCR reaction reagents.

9. A Fusion mutation detection method is characterized by comprising the following steps:

constructing a library required for detection by using the simultaneous RNA and DNA library construction method according to any one of claims 4 to 8;

10. A reverse transcription kit comprising the reverse transcription system of claim 3.

11. The kit for synchronously constructing the RNA and the DNA is characterized by at least comprising a mixed sample formed by quantitatively mixing an RNA sample and a DNA sample, an rhPrimer and an RNase H2 enzyme.