Construction method and application of next-generation sequencing library of PCR (Polymerase Chain Reaction) long amplicons

Abstract

The invention discloses a construction method of a next-generation sequencing library of a PCR (Polymerase Chain Reaction) long amplicon, which comprises the following steps: carrying out PCR amplification on a target sequence by using a primer to obtain the PCR long amplicon; digesting and fragmenting through a mixed enzyme system, and performing terminal repair and A addition, the mixed enzyme system comprising Exo I enzyme, UDG enzyme and Taq DNA polymerase; the A-added product is subjected to sequencing linker connection, and a linker connection product in the form of linker-DNA insertion fragment-linker is obtained; and carrying out a first round of purification treatment to obtain a next-generation sequencing library, which can be directly used on a machine, and also can be used on the machine after being subjected to further PCR amplification and enrichment of the library. According to the method, the PCR long amplicon library for the next-generation sequencing platform can be rapidly constructed, the operation is simple and convenient, the method is suitable for large-scale application, the PCR long amplicon is interrupted by adopting UDG enzyme and dUTP, a specific enzyme digestion recognition site is not needed, and the randomness is better.

Description

Technical Field

[0001] The present invention relates to the technical field of second-generation sequencing, and in particular to a method for constructing a second-generation sequencing library of a PCR long amplicon and an application thereof. Background Art

[0002] Infectious diseases are one of the main causes of human death. There are many types of pathogenic microorganisms, and with climate change, globalization and urbanization, difficult pathogenic microorganisms continue to emerge, and the spread of infectious diseases has also significantly accelerated, posing a great challenge to the global public health system. Traditional pathogen detection technology has obvious limitations, such as single pathogen detection, low accuracy, long detection cycle, and inability to detect emerging pathogens. With the advancement and development of sequencing technology, the second-generation sequencing technology, with its high throughput, high accuracy and low cost, has played an increasingly important role in several global infectious epidemics.

[0003] At present, the application of second-generation sequencing in the direction of pathogens is mainly divided into metagenomic sequencing and targeted sequencing. Targeted sequencing has been increasingly used due to its high sensitivity and low sequencing cost. Targeted sequencing generally uses multiplex PCR or probe hybridization to capture all or specific regions on the genome of a species or a class of species, and then performs sequencing to obtain the full genome sequence and drug resistance gene sequence information of pathogenic microorganisms, intuitively and profoundly revealing the deep information of nucleic acid molecules, and providing strong support for the identification, detection and research of pathogenic microorganisms. However, the application of targeted sequencing on pathogenic microorganisms is still facing many challenges. The workflow of probe hybridization capture is cumbersome, time-consuming, and the library construction is difficult and costly. Multiplex PCR can be divided into two schemes: short amplicon and long amplicon. Short amplicon generally uses 2 rounds of PCR to build a library. The process is simple, but the primer design is difficult, the primer dimer phenomenon cannot be avoided, and there are many types of pathogenic microorganisms, rapid evolution and mutation, and it is easy to cause off-target problems caused by mutations in the primer position. Long amplicon is a scheme that is more tolerant to microbial mutations. Generally, long target fragments are obtained by PCR first, and then the long target fragments are used to construct a DNA library. The library construction generally adopts mechanical interruption, enzymatic interruption or transposase method. The process of mechanical interruption and enzymatic interruption is relatively cumbersome, and mechanical interruption has high requirements on the sample starting amount and sample quality. Enzymatic interruption requires a more complex fragmentation mixed enzyme system, and the cost of transposase is relatively high and has certain sequence preference. The present invention intends to establish a library construction method for PCR long amplicon to simplify the library construction process and reduce costs. Summary of the invention

[0004] The object of the present invention is to provide a method for constructing a second-generation sequencing library of a PCR long amplicon, and the object of the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for constructing a second-generation sequencing library of a PCR long amplicon, comprising the following steps: S1. PCR amplification: using primers to perform PCR amplification on the target sequence to obtain a PCR long amplicon; S2, digesting and fragmenting the PCR long amplicon through a mixed enzyme system, and performing end repair and A addition; the mixed enzyme system includes Exo I enzyme, UDG enzyme and Taq DNA polymerase; S3, connecting the A-added product to a sequencing adapter to obtain an adapter-connected product; S4. Purification of the adapter ligation product: subjecting the adapter ligation product to a first round of purification treatment.

[0005] As some specific embodiments of the present invention, the construction method further comprises the following steps: S5. Library enrichment: The purified adapter-ligated products are used as templates for PCR amplification to enrich the library; S6. Purification of library enrichment products: The library enrichment products are subjected to a second round of purification to obtain a second-generation sequencing library.

[0006] In some specific embodiments of the present invention, in step S1, when PCR amplification is performed, the PCR reaction system includes: primers, dNTPs, DNA polymerase, and template DNA.

[0007] As some specific embodiments of the present invention, the primers are specific primers, including one or more pairs of primers.

[0008] As some specific embodiments of the present invention, the molar ratio of dATP, dCTP, dGTP, dTTP, and dUTP in the dNTPs is 1:1:1:1:0.05-3; As some specific embodiments of the present invention, the DNA polymerase has only 5'→3' DNA polymerase activity and 5'→3' exonuclease activity, but no 3'→5' exonuclease activity, and can be common Taq DNA polymerase, with a dosage of 1-5 U.

[0009] As some specific embodiments of the present invention, in step S1, the reaction procedure of PCR amplification includes: S11: 95°C 2-5 min, 1 cycle; S12: 95℃ 10~30s, 55℃~72℃ 20s~5min, 20~35 cycles; S13: 72°C for 3 min, 1 cycle.

[0010] As some specific embodiments of the present invention, the PCR reaction system also includes UDG enzyme, and the amount of the UDG enzyme is 0.1~2 U. The PCR amplification in step S1 is first incubated at 37°C before the reaction procedures of denaturation, annealing, and extension. The incubation time is 5 minutes. The UDG enzyme is heat-sensitive and easily inactivated at high temperatures. The heat-sensitive UDG enzyme is added to the PCR reaction system and incubated at 37°C in advance to remove possible aerosol contamination of the PCR product. The UDG enzyme is inactivated at the subsequent PCR reaction denaturation temperature.

[0011] As some specific embodiments of the present invention, in step S1, the length of the PCR long amplicon is greater than the maximum read length of the second-generation sequencing, and the length of the PCR long amplicon is 400bp-2000bp.

[0012] In some specific embodiments, a 25 µL reaction system is prepared in a 200 µL PCR tube: 2 µL of a primer mixture containing primers, 5 µL of a PCR reaction buffer containing dNTPs, 0.25-2 µL of a DNA polymerase, 6 µL of a template DNA, dUTP, DMSO, 0.25 µL of a heat-sensitive UDG enzyme may be added, and the volume is made up to 25 µL with sterile water. The primer mixture contains at least two specific primers for amplifying the target sequence, and the concentration of each primer is 0.04-0.8 µmol / L.

[0013] Use 100 mM dATP, 100 mM dCTP, 100 mM dGTP, 100 mM dTTP, and KCl and MgCl 2 The PCR reaction buffer was prepared with Tris HCl buffer; in the prepared PCR reaction buffer, the concentration of Tris HCl was 75-150 mM, the concentration of KCl was 200-500 mM, and the concentration of MgCl was 2 The concentration of dATP is 5~15 mM, the concentrations of dCTP, dGTP, and dTTP are 0.5~2 mM, and the pH value of the PCR reaction buffer is 8.4.

[0014] In the prepared PCR amplification reaction system, the concentrations of dATP, dCTP, dGTP, and dTTP are 0.1~0.4 mM, respectively, and the concentration ratio of dUTP to dTTP is 0.05~3:1. The amount of dUTP is adjusted according to the size of the required library insert fragment, and the ratio of dUTP to dTTP directly affects the size of the amplicon fragment. A certain proportion of dUTP is added to the PCR reaction system, and the ratio of dUTP:dTTP is controlled within the range of 0.05~3, so that the U bases are randomly distributed on the sequence of the PCR amplification product (amplicon).

[0015] As some specific embodiments of the present invention, in step S2, the digestion refers to digesting the primers remaining in the PCR reaction system, and the fragmentation refers to digesting the PCR long amplicon into short fragments. Step S2 specifically includes: adding a digestion enzyme mixture containing Exo I enzyme and UDG enzyme to the PCR amplification reaction product obtained in step S1, and supplementing dATP.

[0016] As some specific embodiments of the present invention, in step S2, the mixed enzyme system includes Exo I enzyme, UDG enzyme, Taq DNA polymerase and dATP. Exo I enzyme can digest single-stranded DNA and remove the primers remaining in the reaction system; UDG enzyme can cleave the N-sugar bond between the uracil base and the sugar-phosphate backbone, eliminate uracil from single- or double-stranded DNA, and break the long amplicon that eliminates uracil into short fragments of 200-500 bp in size under the action of heat; under the action of the Taq enzyme remaining in the PCR system of the previous round (step S1), the repair end is extended from the 5' end to the 3' end on the broken fragment, and A is added to the 3' end.

[0017] As some specific embodiments of the present invention, in step S2, when constructing the reaction system of digestion, fragmentation and end repair plus A, the amount of Exo I enzyme added is 5~40 U, ​​the amount of UDG enzyme added is 0.1~2 U; and dATP is supplemented, and the concentration of dATP in the reaction system is 0.1~0.4 mM.

[0018] As some specific embodiments of the present invention, the reaction procedure of step S2 includes: S21: 37°C for 5-20 min, 1 cycle; S22: 80℃ 10-20 min, 1 cycle; S23: 65℃ for 10-30 min, 1 cycle.

[0019] As some specific embodiments of the present invention, in the above step S21, at 37° C., the Exo I enzyme and the UDG enzyme act, wherein the Exo I enzyme degrades the single-stranded DNA in the direction of 3'→5', thereby degrading the primers remaining in the PCR system; the UDG enzyme cleaves the N-glycosyl bond between the uracil base and the sugar phosphate backbone, eliminating uracil from the PCR product; In step S22, at 80°C, the Exo I enzyme and the UDG enzyme are inactivated, and the long amplicon of uracil is eliminated and broken into short fragments under the action of heat, that is, the fragmentation of the PCR long amplicon is completed; In step S23, at 65°C, Taq enzyme extends and repairs the ends of the broken fragments from the 5' end to the 3' end under the 5'→3' exonuclease activity and 5'→3' polymerase activity, and adds A at the 3' end.

[0020] As some specific embodiments of the present invention, the reaction system of step S3 includes: a ligase, a sequencing adapter, and a reaction product obtained in step S2.

[0021] As some specific embodiments of the present invention, the ligase includes T4 DNA ligase, and the amount of the T4 DNA ligase used is 1000~6000 U.

[0022] As some specific embodiments of the present invention, the sequencing adapter is a universal library adapter of the Illumina sequencing platform, the MGI sequencing platform or the Ion Torrent sequencing platform.

[0023] As some specific embodiments of the present invention, in step S3, the reaction conditions for the linker connection are: 20° C. for 10 to 30 min.

[0024] As some specific embodiments of the present invention, the reaction system in step S3 also includes a ligation reaction buffer, which contains 100-500 mM Tris-HCl, 10-40 mM MgCl 2 , 1~4 mM DTT, 1~4 mM ATP, and 7.5~30% (v / v) PEG 6000.

[0025] As some specific embodiments of the present invention, the step of constructing the reaction system in step S3 specifically includes: adding 5 μL of ligase, 2.5 μL of sequencing adapter, and 25 μL of ligation reaction buffer to the reaction product obtained in step S2, and making up to 100 μL with sterile water. The pH of the Tris-HCl is 7.6 and the temperature is 25 °C.

[0026] The product obtained in step S2 is ligated with a sequencing adapter by T4 DNA ligase; the product with the adapter is the second-generation sequencing library, which can be directly used on the machine after the next purification treatment, or further PCR amplification and enrichment of the library.

[0027] As some specific embodiments of the present invention, in step S3, the adapter ligation product obtained is in the form of "adapter-DNA insert-adapter".

[0028] As some specific embodiments of the present invention, in step S4 and / or step S6, the purification treatment is specifically purification and recovery using DNA purification magnetic beads.

[0029] As some specific embodiments of the present invention, the first round of purification treatment in step S4 includes: mixing the DNA purification magnetic beads and the adapter connection product obtained in step S3 and standing for reaction, removing the supernatant after magnetic adsorption, washing and drying the magnetic beads, and eluting the adapter connection product adsorbed on the magnetic beads with sterile ultrapure water. The product obtained after this step of purification can be used as a second-generation sequencing library, or the library can be further enriched by PCR amplification in the subsequent step.

[0030] As some specific embodiments of the present invention, in step S4, the first round of purification treatment specifically includes the following steps: S41, use DNA purification magnetic beads to balance to room temperature; S42, take out all the adapter ligation products obtained in step S3 into a new centrifuge tube, take 60 µL of DNA purification magnetic beads into the centrifuge tube, mix thoroughly and let it react for 5 min; S43. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 min until the solution is clear and the magnetic beads are completely adsorbed. Use a pipette to carefully discard the supernatant. S44, add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant; S45, repeat step S44 once; S46. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; S47. Add 22 µL of sterile ultrapure water for elution. Use a pipette to gently pipette and mix thoroughly. Place at room temperature for 5 minutes. Centrifuge the tube briefly and place it on a magnetic rack. After the solution becomes clear (about 2 minutes), carefully transfer 20 µL of the supernatant to a new centrifuge tube. Do not touch the magnetic beads.

[0031] As some specific embodiments of the present invention, when performing PCR amplification in step S5, the reaction system includes: a universal library primer pair, a nucleic acid amplification reaction solution, and the purified adapter ligation product obtained in step S4.

[0032] As some specific embodiments of the present invention, the nucleic acid amplification reaction solution includes dNTPs and DNA polymerase, and also includes PCR reaction buffer.

[0033] Prepare a 50 µL reaction system in a 200 µL PCR tube: 5 µL of universal library primer pair, 25 µL of nucleic acid amplification reaction solution, and 20 µL of the purified adapter ligation product obtained in step S4.

[0034] The universal library primer pair is adapted to the sequencing adapter of step S3 according to the selected second-generation sequencing platform, and can be the universal library primer of Illumina sequencing platform, MGI sequencing platform or Ion Torrent sequencing platform.

[0035] As some specific embodiments of the present invention, the PCR amplification procedure for Chinese library enrichment in step S5 is as follows: S51: 95°C 3-8min, 1 cycle; S52: 95°C for 10-30 s, 60°C for 15 s, 72°C for 30 s, 3-15 cycles; S53: 72℃ 3-5min, 1 cycle.

[0036] As some specific embodiments of the present invention, in step S6, the second round of purification treatment includes: mixing the DNA purification magnetic beads with the library enrichment product obtained in step S5 and letting it stand for reaction, removing the supernatant after magnetic adsorption, washing and drying the magnetic beads, and eluting the library enrichment product adsorbed on the magnetic beads with sterile ultrapure water.

[0037] As some specific embodiments of the present invention, in step S6, the second round of purification treatment specifically includes the following steps: S61, DNA purification magnetic beads are equilibrated to room temperature; S62, take 45 µL of DNA purification magnetic beads and add them to the library enrichment PCR product obtained in step S5, mix thoroughly and let it react for 5 min; S63. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 min until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. S64, add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant; S65, repeat step S64 once; S66. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; S67. Add 22 µL of sterile ultrapure water for elution. Use a pipette to gently pipette and mix thoroughly. Place at room temperature for 5 minutes. Centrifuge the tube briefly and place it on a magnetic rack to stand. After the solution becomes clear (about 2 minutes), carefully transfer 20 µL of the supernatant to a new centrifuge tube. Do not touch the magnetic beads.

[0038] In a second aspect, the present invention provides an application of the second-generation sequencing library construction method described in any one of the above items in second-generation sequencing.

[0039] Compared with the prior art, the present invention has the following beneficial effects: (1) The method of the present invention can quickly construct a PCR long amplicon library for the second-generation sequencing platform. It has low reagent cost, simple process, few purification steps, and is suitable for large-scale application.

[0040] (2) Compared with conventional library construction methods, the present invention can simplify the library construction process. In conventional library construction methods, after PCR amplification, the PCR product needs to be purified, homogenized, fragmented, and then end-repaired and A-added. In the present invention, after obtaining the PCR long amplicon, there is no need to purify the PCR product, and the fragmentation and end-repair and A-addition reactions are completed in one step, shortening the library construction process.

[0041] (3) The fragmentation enzyme system of the conventional library construction method usually uses more than two mixed enzyme systems (such as the commercially available NEB product uses a mixture of VVN nuclease and T7 endonuclease), and the end repair and A addition are performed using a combination of 3 to 4 mixed enzymes (usually T4PNK phosphokinase, T4 DNA polymerase, Klenow large fragment, taq DNA polymerase, etc.). The enzyme system is complex and costly. In the present invention, after the primer is digested by Exo I enzyme and uracil is eliminated by UDG enzyme, the phosphate group is still retained. Only the 5'-3' exonuclease activity of taq DNA polymerase and the property of adding an A tail to the 3' end of the blunt end can be used to achieve end repair and A addition, without the need to use expensive enzymes and complex enzyme systems.

[0042] (4) The present invention can also reduce aerosol contamination. Since the PCR long amplicon product does not need to be purified, the library construction process is shortened, and the number of times the cover is opened is reduced, the probability of aerosol contamination is reduced. At the same time, the PCR long amplicon product of the present invention is directly fragmented, and the product does not form contamination. In addition, since the PCR long amplicon product of the present invention contains uracil bases, it can be adapted to a conventional anti-contamination system (UDG enzyme) to further reduce the risk of contamination, that is, a step of incubation at 37°C is added before the PCR reaction starts to remove possible PCR aerosol contamination.

[0043] (5) The present invention uses uracil bases and UDG enzymes, which can not only establish an anti-pollution system, but also digest and fragment PCR long amplicons. A certain proportion of dUTP is incorporated during the first step of PCR amplification, so that uracil bases are randomly incorporated into the obtained PCR long amplicon products, and then the PCR products are fragmented by utilizing the characteristics of UDG enzyme hydrolysis and thermal action to break the uracil glycosidic bond in double-stranded DNA; compared with the traditional fragmentation interruption method, no specific enzyme recognition site is required, so the randomness is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 It is a schematic diagram of the process of constructing a second-generation sequencing library of a PCR long amplicon of the present invention; Figure 2 This is the library fragment distribution diagram of Rubella virus 1EL1 type in Example 1; Figure 3 This is the library fragment distribution diagram of the new coronavirus in Example 2. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0046] like Figure 1 FIG. 1 is a flow chart of the method for constructing a second-generation sequencing library of a PCR long amplicon of the present invention, which specifically includes the following steps: (1) PCR amplification of the target sequence is performed using primers to obtain a long PCR amplicon with a larger fragment length; the amplicon length is greater than the maximum read length of the second-generation sequencing, generally greater than 400 bp, the primers are specific primers, the thermosensitive UDG enzyme is pre-incubated at 37°C to remove possible aerosol contamination of the PCR product, and is inactivated at the subsequent PCR reaction denaturation temperature; a certain proportion of dUTP is added to the PCR reaction system to randomly distribute the PCR amplification product in the sequence; (2) The PCR long amplicon is digested with a mixed enzyme system and the ends are repaired and A is added; the mixed enzyme system includes Exo I enzyme, UDG enzyme, Taq enzyme and dATP. Exo I enzyme can digest single-stranded DNA and remove residual primers in the reaction system. UDG enzyme can cleave the N-glycosyl bond between the uracil base and the sugar phosphate backbone, eliminating uracil from single- or double-stranded DNA. Under the action of heat, the long amplicon with uracil eliminated is broken into fragments of 200-500 bp in size. Under the action of Taq enzyme, the ends of the broken fragments are extended from the 5' end to the 3' end to repair the ends, and A is added to the 3' end. (3) Connecting the A-added product to a sequencing adapter to obtain an adapter-connected product in the form of "adapter-DNA insert-adapter"; adding a sequencing adapter using T4 DNA ligase, wherein the sequencing adapter may be a universal library adapter for an Illumina sequencing platform, an MGI sequencing platform, or an Ion Torrent sequencing platform, depending on the second-generation sequencing platform; the product after adding the adapter is the second-generation sequencing library; (4) Purification of adapter ligation products: The adapter ligation products are subjected to the first round of purification treatment and purified and recovered using DNA purification magnetic beads. After purification and recovery, they can be directly used on the machine or further PCR amplified to enrich the library; (5) Library enrichment: The purified adapter-ligated products are used as templates for PCR amplification to enrich the library; the primers used for library enrichment are universal library amplification primers for each second-generation sequencing platform; (6) Purification of library enrichment products: The library enrichment products are subjected to a second round of purification and recovered using DNA purification magnetic beads to obtain a second-generation sequencing library.

[0047] Example 1 - Whole genome detection of Rubella virus 1EL1 Primer design: Nine pairs of primers were designed based on the reference sequence KT962863.1 (GenBank number) of rubella virus type 1EL1, with amplicon lengths ranging from 900 to 1300 bp, covering the full-length genome of rubella virus, as shown in Table 1 below. Primer pairs 1, 3, 5, 7, and 9 were mixed in the same tube for amplification (RV pool 1), and primer pairs 2, 4, 6, and 8 were mixed in another tube for amplification (RV pool 2).

[0048] Table 1 Rubella virus 1EL1 type primer sequence list

[0049] Experimental process: Rubella virus 1EL1 pseudovirus (reference sequence KT962863.1, synthesized by Beijing Qingke Biotechnology Co., Ltd.) was synthesized, and the nucleic acid of 1EL1 pseudovirus was prepared using the above two primers. The library was repeated three times, and high-throughput sequencing was performed after the library preparation was completed. The obtained sequencing data were assembled and typed to evaluate the library quality.

[0050] (1) Sample preparation The pseudovirus RNA was extracted using a fully automatic nucleic acid extractor (ShuoShi Biotechnology, catalog number: SSNP-9600A) and a nucleic acid extraction kit (magnetic bead method, ShuoShi Biotechnology, catalog number: SDK60105).

[0051] (2) Reverse transcription reaction M-MLV reverse transcriptase was used to reverse transcribe viral RNA to synthesize cDNA. The reverse transcription reaction system is shown in Table 2 below.

[0052] Table 2 Reverse transcription reaction system

[0053] In the above table: a. 10×RT Mix is ​​a reverse transcription buffer, including 250~750 mM Tris-HCl (pH 8.3), 375~900 mM KCl, 15~50 mM MgCl 2 、50~150 mM DTT. In this example, 10×RT Mix includes 500 mM Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl 2 , 100 mM DTT.

[0054] b. M-MLV Reverse Transcriptase (100 U / μL) was purchased from SuoShi Biotechnology, catalog number CSEN-006-4KU.

[0055] c. Random haxamers are 6-base random primers (random N6 primers, NNNNNN), synthesized by Shanghai Bio-Technology Co., Ltd.; d. Nuclease-free water was purchased from Thermo Fisher Scientific.

[0056] The reverse transcription reaction procedure is as follows: Step 1: 25°C for 5 min, 1 cycle; Step 2: 50°C for 45 min, 1 cycle; Step 3: 85°C for 5 min, 1 cycle.

[0057] (3) Multiplex PCR long amplicon reaction: Using the cDNA product from the previous round as a template, a PCR amplification reaction system as shown in Table 3 was prepared: Table 3 PCR amplification reaction system

[0058] In the above table: a. 5×PCR Reaction Buffer (with dNTP) includes 75~150 mM Tris HCl (pH 8.4), 200~500 mM KCl, 5~15 mM MgCl 2 , 0.5~2 mM dATP, 0.5~2 mM dTTP, 0.5~2 mM dCTP, 0.5~2 mM dGTP. In this embodiment, 5×PCR Reaction Buffer (with dNTP) includes 100 mM TrisHCl (pH 8.4), 250 mM KCl, 7.5 mM MgCl 2, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 1 mMdGTP.

[0059] b. Taq DNA polymerase was purchased from ShuoShi Biotechnology, catalog number CSP-003-250U.

[0060] c. DMSO was purchased from Merck Biotechnology; d. dATP, dTTP, dCTP, dGTP, dUTP, UDG, and Nuclease-free water were purchased from Thermo Fisher Scientific.

[0061] The PCR reaction procedure is as follows: Step 1: 37°C for 5 min, 1 cycle; Step 2: 95°C for 5 min, 1 cycle; Step 3: 95°C for 30s, 63°C for 3min, 30 cycles; Step 4: 72°C for 3 min, 1 cycle.

[0062] (4) Digestion and end-repair plus A reaction: Mix the two tubes of PCR products from the previous round, and then add the following enzymes and reagents according to the system shown in Table 4: Table 4 Digestion and end repair plus A reaction system

[0063] The digestion and final addition A reaction procedures are as follows: Step 1: 37°C for 15 min, 1 cycle; Step 2: 80°C for 20 min, 1 cycle; Step 3: 65°C for 15 min, 1 cycle; (5) Linker ligation reaction: In the last round of digestion and the final addition of A product, enzymes and reagents were added according to the reaction system shown in Table 5: Table 5 Linker ligation reaction system

[0064] In the above table: a. Ligatin Buffer is a ligase buffer, including 100~500 mM Tris-HCl, 10~40 mM MgCl 2 , 1-4 mM DTT, 1-4 mM ATP, and 7.5-30% (w / w) PEG 6000, in this example 400 mM Tris-HCl, 40 mM MgCl 2, 4 mM DTT, 4 mM ATP, and 30% (v / v) PEG 6000; b. T4 DNA Ligase was purchased from Yisheng Biotechnology; c. The product name of DNA Adapter X for Illumina is TruSeq DNA UD Indexes (24indexes, 96 samples); d. ddH 2 O is nuclease-free water, purchased from Thermo Fisher Scientific.

[0065] The adapter ligation reaction procedure is as follows: Step 1: 20℃ for 15min, 1 cycle.

[0066] (6) Purification of the linker ligation reaction product: A. DNA purification magnetic beads are balanced to room temperature; B. Take 60 µL of DNA purification magnetic beads and add them to the adapter ligation product in the previous step. Mix thoroughly and let it react for 5 minutes. C. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. D. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant; E. Repeat the previous step once; F. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; G. Add 22 µL of sterile ultrapure water for elution, use a pipette to gently blow and mix thoroughly, and place at room temperature for 5 min. Centrifuge the tube briefly and place it on a magnetic rack to stand. After the solution becomes clear (about 2 min), carefully transfer 20 µL of the supernatant to a new centrifuge tube without touching the magnetic beads.

[0067] (7) Library enrichment reaction: The purified adapter ligation product from the previous round was used as a template to prepare the reaction system shown in Table 6 below: Table 6 Library enrichment reaction system

[0068] In the above table: a. 10 × PCR Reaction Buffer (with dNTP) includes 150~300 mM Tris HCl (pH 8.4), 200~500 mM KCl, 5~15 mM MgCl 2, 0.5-2 mM dATP, 0.5-2 mM dTTP, 0.5-2 mM dCTP, 0.5-2 mM dGTP, in this example, 200 mM Tris HCl (pH 8.4), 500 mM KCl, 15 mM MgCl 2 , 2mM dATP, 2mM dTTP, 2mM dCTP, 2mM dGTP; b. Illumina library primers were synthesized by Sangon Biotechnology. The upstream primer sequence is: 5'-CAAGCAGAAGACGGCAT-3' (SEQ ID NO. 75), The downstream primer sequence is: 5'-AATGATACGGCGACCAC-3' (SEQ ID NO. 76).

[0069] The PCR reaction procedure is as follows: Step 1: 95°C for 5 min, 1 cycle; Step 2: 95°C for 30 s, 60°C for 15 s, 72°C for 30 s, 4 cycles; Step 3: 72°C for 5 min, 1 cycle; (8) Purification of library enrichment products A. DNA purification magnetic beads are balanced to room temperature; B. Take 45 µL of DNA purification magnetic beads and add them to the library enrichment PCR product in the previous step. Mix thoroughly and let react for 5 minutes. C. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. D. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant; E. Repeat the previous step once; F. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; G. Add 22 µL of sterile ultrapure water for elution, use a pipette to gently blow and mix thoroughly, and place at room temperature for 5 min. Centrifuge the tube briefly and place it on a magnetic rack to stand. After the solution becomes clear (about 2 min), carefully transfer 20 µL of the supernatant to a new centrifuge tube without touching the magnetic beads.

[0070] (9) Library quality control: Qseq100 was used to detect the size of the product, and qubit 2.0 was used to check the concentration of the library. Figure 2The library fragments are distributed between 200 and 600 bp, with the main peak at 300 to 350 bp. The peaks are concentrated, which meets the requirements of the machine for fragment size.

[0071] (10) Sequencing The obtained libraries were standardized and mixed in equal amounts, and the mixed libraries were sequenced in parallel using the Illumina NextSeq550Dx sequencing platform and PE150 sequencing type.

[0072] (11) Data analysis The analysis steps include basic steps such as filtering adapter primer sequences, primer excision, alignment, and consensus sequence output.

[0073] Results: The RNA extracted from the Rubella virus 1EL1 pseudovirus was repeated three times for library construction, with an alignment rate of more than 99% and a genome coverage of more than 99.75%. The full-length viral genome was obtained and correctly typed, and the library construction results were good, as shown in Table 7.

[0074] Table 7 Rubella virus 1EL1 pseudovirus library construction results

[0075] Conclusion: The sequencing library constructed by the PCR long amplicon library construction method of the present invention has good library quality and meets the requirements of the machine.

[0076] Example 2 - Whole genome detection of the new coronavirus Primer design: 28 pairs of primers were designed for the reference sequence NC_045512.2 (ACCESSION number) of the new coronavirus, with amplicon lengths between 1000 and 1300 bp, covering the full-length genome of the new coronavirus, as shown in Table 8 below, where primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 were mixed in the same tube for amplification (nCoV pool 1), and primer pairs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 were mixed in another tube for amplification (nCoV pool 2).

[0077] Table 8: New Coronavirus Primer Sequence List

[0078] Experimental process: Synthesize the new coronavirus pseudovirus (reference sequence NC_045512, synthesized by Beijing Qingke Biotechnology), use the above two tubes of primers to prepare the nucleic acid library of the new coronavirus pseudovirus, repeat 3 times, and perform high-throughput sequencing after the library preparation is completed. The obtained sequencing data are assembled and typed to evaluate the library quality.

[0079] (1) Sample preparation The pseudovirus RNA was extracted using a fully automatic nucleic acid extractor (ShuoShi Biotechnology, catalog number: SSNP-9600A) and a nucleic acid extraction kit (magnetic bead method, ShuoShi Biotechnology, catalog number: SDK60105).

[0080] (2) Reverse transcription reaction M-MLV reverse transcriptase was used to reverse transcribe viral RNA to synthesize cDNA. The reaction system is shown in Table 9 below.

[0081] Table 9 Reverse transcription reaction system

[0082] In the above table: a. 10×RT Mix is ​​a reverse transcription buffer, including 250~750 mM Tris-HCl (pH 8.3), 375~900 mM KCl, 15~50 mM MgCl 2 , 50-150 mM DTT. In this example, 10×RT Mix includes 500 mM Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl 2 , 100 mM DTT; b. M-MLV Reverse Transcriptase (100 U / μL) was purchased from ShuoShi Biotechnology, catalog number CSEN-006-4KU; c. Random haxamers are 6-base random primers (random N6 primers, NNNNNN), synthesized by Shanghai Bio-Technology Co., Ltd.; d. Nuclease-free water is nuclease-free water purchased from Thermo Fisher Scientific.

[0083] The reverse transcription reaction procedure is as follows: Step 1: 25°C for 5 min, 1 cycle; Step 2: 50°C for 45 min, 1 cycle; Step 3: 85°C for 5 min, 1 cycle; (3) Multiplex PCR long amplicon reaction: Using the cDNA product from the previous round as a template, a reaction system as shown in Table 10 was prepared: Table 10 PCR amplification reaction system

[0084] In the above table: a. 5×PCR Reaction Buffer (with dNTP) includes 75~150 mM Tris HCl (pH 8.4), 200~500 mM KCl, 5~15 mM MgCl 2 , 0.5-2 mM dATP, 0.5-2 mM dTTP, 0.5-2 mM dCTP, 0.5-2 mM dGTP, in this example, 100 mM Tris HCl (pH 8.4), 250 mM KCl, 7.5 mM MgCl 2 , 1mM dATP, 1mM dTTP, 1mM dCTP, 1mM dGTP; b. Taq DNA polymerase was purchased from ShuoShi Biotechnology, catalog number CSP-003-250U; c. DMSO was purchased from Merck Biotechnology; d. dATP, dTTP, dCTP, dGTP, dUTP, UDG, and Nuclease-free water were purchased from Thermo Fisher Scientific; The PCR reaction procedure is as follows: Step 1: 95°C for 5 min, 1 cycle; Step 2: 95°C for 30s, 63°C for 3min, 30 cycles; Step 3: 72°C for 3 min, 1 cycle.

[0085] (4) Digestion and end-repair plus A reaction: Mix the two tubes of PCR products from the previous round, and then add enzymes and reagents according to the system shown in Table 11: Table 11 Digestion and end repair plus A reaction system

[0086] The digestion and final addition A reaction procedures are as follows: Step 1: 37°C for 15 min, 1 cycle; Step 2: 80°C for 20 min, 1 cycle; Step 3: 65°C for 15 min, 1 cycle; (5) Linker ligation reaction: In the last round of digestion and the final addition of A product, enzymes and reagents were added according to the system shown in Table 12 below: Table 12 Linker ligation reaction system

[0087] In the above table: a. Ligatin Buffer is a ligase buffer, including 100~500 mM Tris-HCl, 10~40 mM MgCl 2 , 1-4 mM DTT, 1-4 mM ATP, and 7.5-30% (v / v) PEG 6000, in this example 400 mM Tris-HCl, 40 mM MgCl 2 , 4 mM DTT, 4 mM ATP, and 30% (v / v) PEG 6000; b. T4 DNA Ligase was purchased from Yisheng Biotechnology; c. DNA Adapter X for Illumina is the TruSeq DNA UD Indexes (24 indexes, 96 samples) product; d. ddH 2 O is nuclease-free water, purchased from Thermo Fisher Scientific.

[0088] The adapter ligation reaction procedure is as follows: Step 1: 20 ℃ for 15 min, 1 cycle.

[0089] (6) Double-selection purification of linker ligation reaction products A. DNA purification magnetic beads are balanced to room temperature; B. Take 50 µL of DNA purification magnetic beads and add them to the library enrichment PCR product in the previous step. Mix thoroughly and let it react for 5 minutes. C. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then use a pipette to carefully transfer the supernatant to a new centrifuge tube. D. Take 40 µL of DNA purification magnetic beads and add them to the supernatant of the previous step. Mix thoroughly and let it react for 5 min. E. Place the centrifuge tube on the magnetic rack and let it stand for 1-2 minutes until the solution is clear and the magnetic beads are completely adsorbed. Then, carefully discard the supernatant with a pipette. F. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant; G. Repeat the previous step once; H. Keep the centrifuge tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads for 5-10 minutes until no ethanol remains; I. Add 22 µL of sterile ultrapure water for elution, use a pipette to gently pipette and mix thoroughly, place at room temperature for 5 min, centrifuge the tube briefly and place it on a magnetic stand to stand, wait for the solution to become clear (about 2 min), carefully transfer 20 µL of the supernatant to a new centrifuge tube, and be careful not to touch the magnetic beads.

[0090] (7) Library quality control: Qseq100 was used to detect the size of the product, and qubit 2.0 was used to check the concentration of the library. Figure 3 The library fragments are distributed between 200 and 600 bp, with the main peak at 400 to 450 bp. The peaks are concentrated, which meets the requirements of the sequencing machine for the fragment size.

[0091] (8) Sequencing The obtained libraries were standardized and mixed in equal amounts, and the mixed libraries were sequenced in parallel using the Illumina NextSeq550Dx sequencing platform and PE150 sequencing type.

[0092] (9) Data analysis The analysis steps include basic steps such as filtering adapter primer sequences, primer excision, alignment, and consensus sequence output.

[0093] Results: The RNA extracted from the new coronavirus pseudovirus was repeated three times for library construction, with an alignment rate of over 99% and a genome coverage of over 99.5%. The full-length viral genome was obtained and correctly typed, and the library construction results were good, as shown in Table 13 below.

[0094] Table 13. Results of the novel coronavirus database construction

[0095] Conclusion: The PCR long amplicon library construction method has a simple process and can remove aerosol contamination caused by PCR amplification. The library quality is good and meets the requirements of the machine.

[0096] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for constructing a second-generation sequencing library of a PCR long amplicon, characterized in that: The steps include: S1. PCR amplification: using primers to perform PCR amplification on the target sequence to obtain a PCR long amplicon; S2, digesting and fragmenting the PCR long amplicon through a mixed enzyme system, and performing end repair and A addition; the mixed enzyme system includes Exo I enzyme, UDG enzyme and Taq DNA polymerase; S3, connecting the A-added product to a sequencing adapter to obtain an adapter-connected product; S4. Purification of the adapter ligation product: subjecting the adapter ligation product to a first round of purification treatment.

2. The method for constructing a second-generation sequencing library according to claim 1, characterized in that: In step S1, when PCR amplification is performed, the PCR reaction system includes: primers, dNTPs, DNA polymerase, and template DNA; The molar ratio of dATP, dCTP, dGTP, dTTP and dUTP in the dNTPs is 1:1:1:1:0.05-3; The DNA polymerase is Taq DNA polymerase, and the dosage of the Taq DNA polymerase is 1-5 U.

3. The method for constructing a second-generation sequencing library according to claim 2, characterized in that: In step S1, the reaction procedure of PCR amplification includes: S11: 95°C 2-5 min, 1 cycle; S12: 95℃ 10~30s, 55℃~72℃ 20s~5min, 20~35 cycles; S13: 72°C for 3 min, 1 cycle.

4. The method for constructing a second-generation sequencing library according to claim 2 or 3, characterized in that: The PCR reaction system also includes UDG enzyme; the dosage of the UDG enzyme is 0.1-2 U; Incubate at 37°C before denaturation, annealing and extension of the PCR amplification reaction.

5. The method for constructing a second-generation sequencing library according to claim 1, characterized in that: In step S2, when constructing the reaction system for digestion, fragmentation, and end repair plus A, the amount of Exo I enzyme added is 5-40 U, ​​and the amount of UDG enzyme added is 0.1-2 U; and dATP is supplemented, and the concentration of dATP in the reaction system is 0.1-0.4 mM; And / or, the reaction procedure of step S2 comprises: S21: 37°C for 5-20 min, 1 cycle; S22: 80℃ 10-20 min, 1 cycle; S23: 65℃ for 10-30 min, 1 cycle.

6. The method for constructing a second-generation sequencing library according to claim 1, characterized in that: The reaction system of step S3 includes: a ligase, a sequencing adapter, and the reaction product obtained in step S2; the ligase includes T4 DNA ligase, and the amount of the T4 DNA ligase is 1000-6000 U; And / or, the reaction conditions for linker connection in step S3 are: reaction at 20° C. for 10 to 30 min.

7. The method for constructing a second-generation sequencing library according to claim 6, characterized in that: The reaction system of step S3 also includes a ligation reaction buffer, which contains 100-500 mM Tris-HCl, 10-40 mM MgCl2, 1-4 mM DTT, 1-4 mM ATP, and 7.5-30% PEG 6000.

8. The method for constructing a second-generation sequencing library according to claim 1, characterized in that: The construction method also includes the following steps: S5. Library enrichment: The purified adapter-ligated products are used as templates for PCR amplification to enrich the library; S6. Purification of library enrichment products: The library enrichment product was subjected to a second round of purification to obtain a next-generation sequencing library.

9. The method for constructing a second-generation sequencing library according to claim 8, characterized in that: In step S5, the reaction system for library enrichment PCR amplification includes: a universal library primer pair, a nucleic acid amplification reaction solution, and the purified adapter ligation product obtained in step S4; the nucleic acid amplification reaction solution includes dNTPs and DNA polymerase.

10. Use of the method for constructing a second-generation sequencing library as described in any one of claims 1 to 9 in second-generation sequencing.

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