A method for constructing and applying next-generation sequencing libraries for PCR long amplicon sequencing.

Abstract

This invention discloses a method for constructing a next-generation sequencing library using long PCR amplicon sequences, comprising: performing PCR amplification on a target sequence using primers to obtain long PCR amplicon sequences; digesting and fragmenting the sequences using a mixed enzyme system, followed by end-repair and A addition, wherein the mixed enzyme system includes Exo I enzyme, UDG enzyme, and Taq DNA polymerase; ligating the A-added product to sequencing adapters to obtain adapter ligation products in the form of "adapter-DNA insert-adapter"; and performing a first round of purification to obtain a next-generation sequencing library, which can be used directly for sequencing or enriched by further PCR amplification before sequencing. This invention enables rapid construction of long PCR amplicon libraries for next-generation sequencing platforms, is simple to operate, and is suitable for large-scale applications. It uses UDG enzyme and dUTP to fragment the long PCR amplicon sequences, eliminating the need for specific restriction enzyme recognition sites and improving randomness.

Description

Technical Field

[0001] This invention relates to the field of next-generation sequencing technology, and more specifically, to a method for constructing and applying next-generation sequencing libraries using PCR long amplicon sequencing. Background Technology

[0002] Infectious diseases are a leading cause of death worldwide. Pathogenic microorganisms are diverse, and with climate change, globalization, and urbanization, increasingly difficult-to-identify pathogens are emerging, significantly accelerating the spread of infectious diseases and posing a major challenge to global public health systems. Traditional pathogen detection techniques have significant limitations, such as limited detection capabilities, low accuracy, long detection cycles, and inability to detect emerging pathogens. With advancements in sequencing technology, next-generation sequencing (NGS) technology, with its high throughput, high accuracy, and low cost, has played an increasingly important role in several global infectious disease outbreaks.

[0003] Currently, the applications of next-generation sequencing in pathogen detection are mainly divided into metagenomic sequencing and targeted sequencing. Targeted sequencing, due to its high sensitivity and low cost, is gaining increasing popularity. Targeted sequencing typically involves capturing all or specific regions of the genome of a species or class of species using multiplex PCR or probe hybridization, followed by sequencing. This allows for the acquisition of the complete genome sequence and drug resistance gene sequences of pathogenic microorganisms, providing a direct and profound understanding of the deeper information in nucleic acid molecules and offering strong support for the identification, detection, and research of pathogenic microorganisms. However, the application of targeted sequencing in pathogenic microorganisms currently faces many challenges. Probe hybridization capture is cumbersome, time-consuming, and library construction is difficult and costly. Multiplex PCR can be divided into short amplicon and long amplicon schemes. Short amplicon sequencing generally uses two rounds of PCR library construction, which is simple, but primer design is difficult, primer dimerization is unavoidable, and pathogenic microorganisms are diverse and rapidly evolving, easily leading to off-target problems due to primer position mutations. Long amplicon sequencing is a more resistant to microbial mutations. It typically involves first obtaining a long target fragment via PCR, and then using this long target fragment to construct a DNA library. Library construction generally employs mechanical fragmentation, enzymatic fragmentation, or transposase methods. Mechanical and enzymatic fragmentation are relatively cumbersome, and mechanical fragmentation requires high sample volume and quality. Enzymatic fragmentation requires a complex fragmentation mixing enzyme system, and transposase methods are relatively expensive and have some sequence bias. This invention aims to establish a method for constructing a long amplicon PCR library, simplifying the library construction process and reducing costs. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a second-generation sequencing library for PCR long amplicon sequencing. This purpose is achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides a method for constructing a next-generation sequencing library for PCR long amplicon sequencing, comprising the following steps:

[0006] S1. PCR amplification: PCR amplification of the target sequence is performed using primers to obtain long PCR amplicons;

[0007] S2. The long PCR amplicon is digested and fragmented using a mixed enzyme system, and then end-repaired with A. The mixed enzyme system includes Exo I enzyme, UDG enzyme, and Taq DNA polymerase.

[0008] S3. The product with added A is ligated into a sequencing adapter to obtain the adapter ligation product.

[0009] S4. Purify the adapter ligation product: Perform the first round of purification on the adapter ligation product.

[0010] As some specific embodiments of the present invention, the construction method further includes the following steps:

[0011] S5. Library enrichment: The purified adapter ligation product is used as a template for PCR amplification to enrich the library.

[0012] S6. Purify the library enrichment product: Perform a second round of purification on the library enrichment product to obtain the next-generation sequencing library.

[0013] In some specific embodiments of the present invention, in step S1, when performing PCR amplification, the PCR reaction system includes: primers, dNTPs, DNA polymerase, and template DNA.

[0014] As some specific embodiments of the present invention, the primers are specific primers, including one or more pairs of primers.

[0015] 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;

[0016] 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 ordinary Taq DNA polymerase, with a dosage of 1~5 U.

[0017] As some specific embodiments of the present invention, the PCR amplification reaction procedure in step S1 includes:

[0018] S11: 95℃ for 2~5 minutes, 1 cycle;

[0019] S12: 95℃ for 10~30s, 55℃~72℃ for 20s~5min, 20~35 cycles;

[0020] S13: 72℃ for 3 minutes, 1 cycle.

[0021] As some specific embodiments of the present invention, the PCR reaction system further includes UDG enzyme, and the amount of UDG enzyme used is 0.1~2 U. In step S1, the PCR amplification is first incubated at 37°C before the denaturation, annealing, and extension reaction procedures. The incubation time is 5 min. The UDG enzyme is heat-sensitive and easily inactivated at high temperatures. Adding heat-sensitive UDG enzyme to the PCR reaction system and pre-incubating at 37°C removes possible PCR product aerosol contamination. The UDG enzyme is inactivated at the denaturation temperature of the subsequent PCR reaction.

[0022] 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.

[0023] In some specific embodiments, a 25 µL reaction system is prepared in a 200 µL PCR tube: 2 µL primer mixture containing primers, 5 µL PCR reaction buffer containing dNTPs, 0.25–2 µL DNA polymerase, 6 µL template DNA, dUTP, DMSO, and optionally 0.25 µL of heat-sensitive UDG enzyme, and then brought to 25 µL with sterile water. The primer mixture contains at least two specific primers for amplifying the target sequence, each primer having a concentration of 0.04–0.8 µmol / L.

[0024] Prepare the PCR reaction buffer using 100 mM dATP, 100 mM dCTP, 100 mM dGTP, 100 mM dTTP, and Tris HCl buffer containing KCl and MgCl2. In the prepared PCR reaction buffer, the concentration of Tris HCl is 75-150 mM, the concentration of KCl is 200-500 mM, the concentration of MgCl2 is 5-15 mM, the concentrations of dATP, dCTP, dGTP, and dTTP are 0.5-2 mM, and the pH of the PCR reaction buffer is 8.4.

[0025] In the prepared PCR amplification reaction system, the concentrations of dATP, dCTP, dGTP, and dTTP are 0.1–0.4 mM, and the concentration ratio of dUTP to dTTP is 0.05–3:1. The amount of dUTP is adjusted according to the desired size of the library insert fragment, and the ratio of dUTP to dTTP directly affects the size of the amplicon fragment. Adding a certain proportion of dUTP to the PCR reaction system and controlling the dUTP:dTTP ratio within the range of 0.05–3 ensures that the PCR amplification product (amplicon) has a random distribution of U bases in its sequence.

[0026] 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 long PCR amplicons into short fragments. Step S2 specifically includes: adding a digestive enzyme mixture containing Exo I enzyme and UDG enzyme to the PCR amplification reaction product obtained in step S1, and supplementing dATP.

[0027] 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 residual primers in the reaction system; UDG enzyme can cleave the N-glycosyl bonds between uracil bases and the sugar-phosphate backbone, eliminating uracil from single or double-stranded DNA, and breaking long amplicones that eliminate uracil under heat into short fragments of 200-500 bp in size; under the action of Taq enzyme remaining in the previous round (step S1) PCR system, the broken fragments are extended from the 5' end to the 3' end to repair the ends, and an A is added to the 3' end.

[0028] As some specific embodiments of the present invention, in step S2, when constructing the reaction system for digestion, fragmentation, and terminal repair with 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, with the concentration of dATP in the reaction system being 0.1~0.4 mM.

[0029] As some specific embodiments of the present invention, the reaction procedure of step S2 includes:

[0030] S21: 37℃ for 5~20 min, 1 cycle;

[0031] S22: 80℃ for 10~20 min, 1 cycle;

[0032] S23: 65℃ for 10~30 min, 1 cycle.

[0033] As some specific embodiments of the present invention, in step S21 above, at 37°C, Exo I enzyme and UDG enzyme function, wherein Exo I enzyme degrades single-stranded DNA in the 3'→5' direction, thereby degrading the primers remaining in the PCR system; UDG enzyme cleaves the N-glycosyl bonds between uracil bases and the sugar phosphate backbone, eliminating uracil from the PCR product;

[0034] In step S22, at 80℃, Exo I enzyme and UDG enzyme are inactivated, and at the same time, the long amplicon of uracil is broken into short fragments under heat, thus completing the fragmentation of the long amplicon of PCR.

[0035] In step S23, at 65°C, Taq enzyme, with its 5'→3' exonuclease activity and 5'→3' polymerase activity, extends and repairs the broken fragment from the 5' end to the 3' end, and adds an A to the 3' end.

[0036] As some specific embodiments of the present invention, the reaction system of step S3 includes: ligase, sequencing adapter, and the reaction product obtained in step S2.

[0037] As some specific embodiments of the present invention, the ligase includes T4 DNA ligase, and the amount of T4 DNA ligase used is 1000~6000 U.

[0038] As some specific embodiments of the present invention, the sequencing adapter is a universal library adapter for the Illumina sequencing platform, MGI sequencing platform, or Ion Torrent sequencing platform.

[0039] As some specific embodiments of the present invention, in step S3, the reaction conditions for the connector connection are: 20°C for 10~30 min.

[0040] As some specific embodiments of the present invention, the reaction system in step S3 further 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% (v / v) PEG 6000.

[0041] As some specific embodiments of the present invention, step S3, which involves constructing the reaction system, 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 then adding sterile water to bring the volume to 100 µL. The Tris-HCl has a pH of 7.6 and a temperature of 25°C.

[0042] The product obtained in step S2 is given a sequencing adapter by T4 DNA ligase; the product with the adapter is the next-generation sequencing library, which can be used directly for sequencing after further purification, or it can be enriched by PCR amplification.

[0043] As some specific embodiments of the present invention, in step S3, the obtained adapter ligation product is in the form of "adapter-DNA insert fragment-adapter".

[0044] As some specific embodiments of the present invention, in step S4 and / or step S6, the purification process specifically involves purification and recovery using DNA purification magnetic beads.

[0045] As some specific embodiments of the present invention, the first round of purification in step S4 includes: mixing the DNA purification magnetic beads with the adapter ligation product obtained in step S3 and allowing them to stand for reaction; removing the supernatant after magnetic adsorption; washing and drying the magnetic beads; and then eluting the adapter ligation product adsorbed on the magnetic beads with sterile ultrapure water. The product obtained after this purification step can be used as a next-generation sequencing library, or it can be further enriched into a library by PCR amplification in subsequent steps.

[0046] As some specific embodiments of the present invention, step S4, the first round of purification treatment specifically includes the following steps:

[0047] S41. Equilibrate to room temperature using DNA purification magnetic beads;

[0048] S42. Take all the adapter ligation products obtained in step S3 into a new centrifuge tube, add 60µL of DNA purification magnetic beads into the centrifuge tube, mix thoroughly, and let stand for 5 min.

[0049] S43. 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.

[0050] S44. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant;

[0051] S45. Repeat step S44 once;

[0052] S46. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0053] S47. Add 22 µL of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µL of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0054] As some specific embodiments of the present invention, the reaction system for PCR amplification in step S5 includes: universal library primer pairs, nucleic acid amplification reaction solution, and the purified adapter ligation product obtained in step S4.

[0055] As some specific embodiments of the present invention, the nucleic acid amplification reaction solution includes dNTPs and DNA polymerase. It also includes PCR reaction buffer.

[0056] Prepare a 50 µL reaction system in a 200 µL PCR tube: 5 µL universal library primer pair, 25 µL nucleic acid amplification reaction solution, and 20 µL of purified adapter ligation product obtained in step S4.

[0057] The universal library primer pair is adapted to the sequencing adapter in step S3, depending on the selected next-generation sequencing platform. It can be a universal library primer for the Illumina sequencing platform, the MGI sequencing platform, or the Ion Torrent sequencing platform.

[0058] As some specific embodiments of the present invention, the PCR amplification procedure for Chinese library enrichment in step S5 is as follows:

[0059] S51: 95℃ for 3~8min, 1 cycle;

[0060] S52: 95℃ for 10~30s, 60℃ for 15s, 72℃ for 30s, 3~15 cycles;

[0061] S53: 72℃ for 3~5 minutes, 1 cycle.

[0062] 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 allowing them to stand for reaction; removing the supernatant after magnetic adsorption; washing and drying the magnetic beads; and then using sterile ultrapure water to elute the library enrichment product adsorbed on the magnetic beads.

[0063] As some specific embodiments of the present invention, step S6, the second round of purification treatment specifically includes the following steps:

[0064] S61. Equilibrate the DNA purification magnetic beads to room temperature.

[0065] 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 stand for 5 min.

[0066] S63. 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.

[0067] S64. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant;

[0068] S65. Repeat step S64 once;

[0069] S66. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0070] S67. Add 22 µL of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µL of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0071] Secondly, the present invention provides an application of the second-generation sequencing library construction method described in any of the above claims in second-generation sequencing.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] (1) The method of the present invention can quickly construct PCR long amplicon libraries for next-generation sequencing platforms. It has low reagent cost, simple process, few purification steps, and is suitable for large-scale application.

[0074] (2) Compared with conventional library construction methods, the present invention simplifies the library construction process. Conventional library construction methods require purification, homogenization, fragmentation, and end repair with A after PCR amplification. The present invention eliminates the need for PCR product purification after obtaining long PCR amplicon, and completes the fragmentation and end repair with A reactions in one step, thus shortening the library construction process.

[0075] (3) Conventional library construction methods typically use two or more mixed enzyme systems for fragmentation (such as commercially available NEB products which use a mixture of VVN nuclease and T7 endonuclease), and end repair and A tail addition use a combination of three to four mixed enzymes (usually T4PNK phosphokinase, T4 DNA polymerase, Klenow large fragment, Taq DNA polymerase, etc.). The enzyme system is complex and costly. In contrast, after the primers are digested by Exo I enzyme and uracil is eliminated by UDG enzyme, the phosphate group is still retained. By utilizing the 5'-3' exonuclease activity of Taq DNA polymerase and its ability to add an A tail at the 3' end of the blunt end, end repair and A tail addition can be achieved without the use of expensive enzymes and complex enzyme systems.

[0076] (4) The present invention can also reduce aerosol contamination. Since the PCR long amplicon product does not need to be purified and the library construction process is shortened, the number of times the lid is opened is reduced, thus reducing the probability of aerosol contamination. 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 carries uracil bases, it can be adapted to conventional anti-contamination system (UDG enzyme) to further reduce the risk of contamination. That is, an incubation at 37°C is added before the start of the PCR reaction to remove possible PCR aerosol contamination.

[0077] (5) This invention uses uracil bases and UDG enzyme, which can not only establish an anti-contamination system, but also digest and fragment long PCR amplicon. A certain proportion of dUTP is incorporated during the first step of PCR amplification, thereby randomly incorporating uracil bases into the obtained long PCR amplicon product. Then, the PCR product is fragmented by utilizing the characteristics of UDG enzyme to hydrolyze and break the uracil glycosidic bonds in the double-stranded DNA and the thermal effect. Compared with the traditional fragmentation method, it does not require specific enzyme recognition sites, so the randomness is better. Attached Figure Description

[0078] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0079] Figure 1 This is a schematic flowchart of the next-generation sequencing library construction method for PCR long amplicon sequencing of the present invention.

[0080] Figure 2 This is a distribution diagram of the rubella virus 1EL1 library fragments in Example 1;

[0081] Figure 3 This is a distribution diagram of the library fragments of the novel coronavirus in Example 2. Detailed Implementation

[0082] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0083] like Figure 1 The diagram shown is a flowchart illustrating the next-generation sequencing library construction method for PCR long amplicon sequencing according to the present invention, which specifically includes the following steps:

[0084] (1) Use primers to amplify the target sequence by PCR to obtain a long PCR amplicon with a larger fragment length; the length of the amplicon is greater than the maximum read length of second-generation sequencing, generally greater than 400 bp, the primers are specific primers, the heat-sensitive UDG enzyme is pre-incubated at 37°C to remove possible PCR product aerosol contamination, and inactivated at the denaturing temperature of the subsequent PCR reaction; a certain proportion of dUTP is added to the PCR reaction system to make the PCR amplification product sequence randomly distributed;

[0085] (2) The long PCR amplicon was digested by a mixed enzyme system and the ends were repaired by adding A. The mixed enzyme system included Exo I enzyme, UDG enzyme, Taq enzyme 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-glycosyl bonds between the uracil base and the sugar phosphate backbone to remove uracil from single or double-stranded DNA. The long amplicon that has removed uracil under heat is broken into fragments of 200-500 bp. 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.

[0086] (3) The product with A is ligated with a sequencing adapter to obtain a "adapter-DNA insert fragment-adapter" type adapter ligation product; a sequencing adapter is added using T4 DNA ligase. The sequencing adapter can be a universal library adapter for the Illumina sequencing platform, MGI sequencing platform or Ion Torrent sequencing platform, depending on the second-generation sequencing platform; the product with the adapter is the second-generation sequencing library.

[0087] (4) Purification of adapter ligation products: The adapter ligation products are purified in the first round and recovered by DNA purification magnetic beads. After purification and recovery, they can be used directly for instrumentation or further PCR amplification to enrich the library.

[0088] (5) Library enrichment: The purified adapter ligation product was used as a template for PCR amplification to enrich the library; the primers for library enrichment were universal library amplification primers for each second-generation sequencing platform.

[0089] (6) Purification of library enrichment products: The library enrichment products are subjected to a second round of purification and purified and recovered using DNA purification magnetic beads to obtain the next-generation sequencing library.

[0090] Example 1 - Whole genome detection of rubella virus 1EL1

[0091] Primer design: Nine primer pairs were designed for the reference sequence KT962863.1 (GenBank number) of rubella virus 1EL1. The amplicon length was between 900 and 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).

[0092] Table 1 Primer sequence list for rubella virus 1EL1

[0093]

[0094] Experimental procedure: Rubella virus 1EL1 pseudovirus (reference sequence KT962863.1, synthesized by Beijing Qingke Biotechnology Co., Ltd.) was synthesized. The nucleic acid of 1EL1 pseudovirus was prepared into a library using the above two primer tubes, repeated three times. After the library preparation was completed, high-throughput sequencing was performed. The obtained sequencing data were assembled and genotyped to evaluate the library quality.

[0095] (1) Sample preparation

[0096] Pseudovirus RNA was extracted using a fully automated nucleic acid extractor (ShuoShi Biotechnology, catalog number: SSNP-9600A) and a nucleic acid extraction kit (magnetic bead method, ShuoShi Biotechnology, catalog number: SDK60105).

[0097] (2) Reverse transcription reaction

[0098] 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.

[0099] Table 2 Reverse Transcription Reaction System

[0100]

[0101] In the above table:

[0102] a. The 10×RT Mix is ​​a reverse transcription buffer, comprising 250–750 mM Tris-HCl (pH 8.3), 375–900 mM KCl, 15–50 mM MgCl2, and 50–150 mM DTT. In this example, the 10×RT Mix comprises 500 mM Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl2, and 100 mM DTT.

[0103] b. M-MLV Reverse Transcriptase (100 U / μL) was purchased from ShuoShi Biotechnology, catalog number CSEN-006-4KU.

[0104] c. Random haxamers are 6-base random primers (random N6 primers, NNNNNN), synthesized by Shanghai Sangon Biotech.

[0105] d. Nuclease-free water was purchased from Thermo Fisher Scientific.

[0106] The reverse transcription procedure is as follows:

[0107] Step 1: 25℃ for 5 minutes, 1 cycle;

[0108] Step 2: 50℃ for 45 minutes, 1 cycle;

[0109] Step 3: 85℃ for 5 minutes, 1 cycle.

[0110] (3) Multiplex PCR long amplicon reaction:

[0111] Using the cDNA product from the previous round as a template, prepare the PCR amplification reaction system as shown in Table 3 below:

[0112] Table 3 PCR amplification reaction system

[0113]

[0114] In the above table:

[0115] a. The 5×PCR Reaction Buffer (with dNTPs) comprises 75–150 mM Tris HCl (pH 8.4), 200–500 mM KCl, 5–15 mM MgCl2, 0.5–2 mM dATP, 0.5–2 mM dTTP, 0.5–2 mM dCTP, and 0.5–2 mM dGTP. In this embodiment, the 5×PCR Reaction Buffer (with dNTPs) comprises 100 mM Tris HCl (pH 8.4), 250 mM KCl, 7.5 mM MgCl2, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, and 1 mM dGTP.

[0116] b. Taq DNA polymerase, purchased from ShuoShi Biotechnology, catalog number CSP-003-250U.

[0117] c. DMSO was purchased from Merck Biotechnology;

[0118] d. dATP, dTTP, dCTP, dGTP, dUTP, UDG, and Nuclease-free water were purchased from Thermo Fisher Scientific.

[0119] The PCR reaction procedure is as follows:

[0120] Step 1: 37℃ for 5 minutes, 1 cycle;

[0121] Step 2: 95℃ for 5 minutes, 1 cycle;

[0122] Step 3: 95℃ for 30 seconds, 63℃ for 3 minutes, 30 cycles;

[0123] Step 4: 72℃ for 3 minutes, 1 cycle.

[0124] (4) Digestion and terminal repair plus A reaction:

[0125] Mix the two tubes of PCR products from the previous round, and then add the enzymes and reagents as shown in Table 4 below:

[0126] Table 4 Digestion and Terminal Repair Plus A Reaction System

[0127]

[0128] The digestion and final addition of A reaction procedure are as follows:

[0129] Step 1: 37℃ for 15 minutes, 1 cycle;

[0130] Step 2: 80℃ for 20 minutes, 1 cycle;

[0131] Step 3: 65℃ for 15 minutes, 1 cycle;

[0132] (5) Joint connection reaction:

[0133] In the previous round of digestion and final addition of product A, enzymes and reagents were added to the reaction system as shown in Table 5 below:

[0134] Table 5. Joint Connection Reaction System

[0135]

[0136] In the above table:

[0137] a. Ligatin Buffer is a ligase buffer comprising 100–500 mM Tris-HCl, 10–40 mM MgCl2, 1–4 mM DTT, 1–4 mM ATP, and 7.5–30% (w / w) PEG 6000. In this example, it comprises 400 mM Tris-HCl, 40 mM MgCl2, 4 mM DTT, 4 mM ATP, and 30% (v / v) PEG 6000.

[0138] b. T4 DNA Ligase was purchased from Yisheng Biotechnology;

[0139] c. The DNA Adapter X for Illumina is named TruSeq DNA UD Indexes (24 indexes, 96 samples);

[0140] d. ddH2O is nuclease-free water, purchased from Thermo Fisher Scientific.

[0141] The connector connection reaction procedure is as follows:

[0142] Step 1: 20℃ for 15 minutes, 1 cycle.

[0143] (6) Purify the reaction products of the connector connection:

[0144] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0145] B. Add 60µL of DNA purification magnetic beads to the adapter ligation product from the previous step, mix thoroughly, and let stand for 5 minutes.

[0146] 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.

[0147] D. Add 500µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30s, and then discard the supernatant;

[0148] E. Repeat the previous step once;

[0149] F. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0150] G. Add 22 µL of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µL of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0151] (7) Library enrichment reaction:

[0152] Using the purified linker ligation product from the previous round as a template, the reaction system shown in Table 6 below was prepared:

[0153] Table 6 Library enrichment reaction system

[0154]

[0155] In the above table:

[0156] a. 10 × PCR Reaction Buffer (with dNTPs) comprises 150–300 mM Tris HCl (pH 8.4), 200–500 mM KCl, 5–15 mM MgCl2, 0.5–2 mM dATP, 0.5–2 mM dTTP, 0.5–2 mM dCTP, and 0.5–2 mM dGTP. In this embodiment, it comprises 200 mM Tris HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2, 2 mM dATP, 2 mM dTTP, 2 mM dCTP, and 2 mM dGTP.

[0157] b. Illumina library primers were synthesized by Sangon Biotech.

[0158] The upstream primer sequence is: 5'-CAAGCAGAAGACGGCAT-3' (SEQ ID NO.75).

[0159] The downstream primer sequence is: 5'-AATGATACGGCGACCAC-3' (SEQ ID NO.76).

[0160] The PCR reaction procedure is as follows:

[0161] Step 1: 95℃ for 5 minutes, 1 cycle;

[0162] Step 2: 95℃ for 30s, 60℃ for 15s, 72℃ for 30s, 4 cycles;

[0163] Step 3: 72℃ for 5 minutes, 1 cycle;

[0164] (8) Purification of library enrichment products

[0165] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0166] B. Add 45µL of DNA purification magnetic beads to the library enrichment PCR product from the previous step, mix thoroughly, and let stand for 5 minutes.

[0167] 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.

[0168] D. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 seconds, and then discard the supernatant;

[0169] E. Repeat the previous step once;

[0170] F. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0171] G. Add 22 µL of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µL of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0172] (9) Document quality control:

[0173] Qseq100 was used to detect the size of the products, and qubit 2.0 was used for library quality control to determine the concentration. The distribution of the library fragments is as follows: Figure 2 As shown, the library fragments are distributed between 200 and 600 bp, with the main peak at 300 to 350 bp. The peak shape is concentrated, which meets the requirements for fragment size for computer testing.

[0174] (10) Sequencing

[0175] The obtained libraries were standardized and mixed in equal amounts. The resulting libraries were then subjected to parallel sequencing using the Illumina NextSeq550Dx sequencing platform and the PE150 sequencing type.

[0176] (11) Data Analysis

[0177] The analysis steps include basic steps such as filtering adapter primer sequences, primer excision, alignment, and output of consistent sequences.

[0178] Results: The RNA extracted from the rubella virus 1EL1 pseudovirus was used for library construction three times. The alignment rate was over 99% and the genome coverage was over 99.75%. The full-length viral genome was obtained and correctly genotyped in all cases. The library construction results were good, as shown in Table 7 below.

[0179] Table 7. Results of Library Construction for Rubella Virus 1EL1 Pseudovirus

[0180]

[0181] Conclusion: The sequencing library constructed using the PCR long amplicon library construction method of this invention has good library quality and meets the requirements for sequencing.

[0182] Example 2 - Whole genome detection of SARS-CoV-2

[0183] Primer design: 28 primer pairs were designed targeting the reference sequence NC_045512.2 (ACCESSION number) of the novel coronavirus. The amplicon length was between 1000 and 1300 bp, covering the full-length genome of the novel coronavirus, as shown in Table 8 below. Primer pairs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 were mixed in one tube for amplification (nCoV pool 1), while 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).

[0184] Table 8 Primer sequence list for SARS-CoV-2

[0185]

[0186] Experimental procedure: Synthesize SARS-CoV-2 pseudovirus (reference sequence NC_045512, synthesized by Beijing Qingke Biotechnology), use the above two primer tubes to prepare a library of SARS-CoV-2 pseudovirus nucleic acid, repeat 3 times, perform high-throughput sequencing after library preparation, assemble and genotype the obtained sequencing data, and evaluate the library quality.

[0187] (1) Sample preparation

[0188] Pseudovirus RNA was extracted using a fully automated nucleic acid extractor (ShuoShi Biotechnology, catalog number: SSNP-9600A) and a nucleic acid extraction kit (magnetic bead method, ShuoShi Biotechnology, catalog number: SDK60105).

[0189] (2) Reverse transcription reaction

[0190] The viral RNA was reverse transcribed using M-MLV reverse transcriptase to synthesize cDNA. The reaction system is shown in Table 9 below.

[0191] Table 9 Reverse Transcription Reaction System

[0192]

[0193] In the above table:

[0194] a. 10×RT Mix is ​​a reverse transcription buffer, comprising 250–750 mM Tris-HCl (pH 8.3), 375–900 mM KCl, 15–50 mM MgCl2, and 50–150 mM DTT. In this embodiment, 10×RT Mix comprises 500 mM Tris-HCl (pH 8.3), 750 mM KCl, 30 mM MgCl2, and 100 mM DTT.

[0195] b. M-MLV Reverse Transcriptase (100 U / μL) was purchased from ShuoShi Biotechnology, catalog number CSEN-006-4KU;

[0196] c. Random haxamers are 6-base random primers (random N6 primers, NNNNNN), synthesized by Shanghai Sangon Biotech.

[0197] d. Nuclease-free water is water without nuclease, purchased from Thermo Fisher Scientific.

[0198] The reverse transcription procedure is as follows:

[0199] Step 1: 25℃ for 5 minutes, 1 cycle;

[0200] Step 2: 50℃ for 45 minutes, 1 cycle;

[0201] Step 3: 85℃ for 5 minutes, 1 cycle;

[0202] (3) Multiplex PCR long amplicon reaction:

[0203] Using the cDNA product from the previous round as a template, prepare the reaction system shown in Table 10 below:

[0204] Table 10 PCR amplification reaction system

[0205]

[0206] In the above table:

[0207] a. The 5×PCR Reaction Buffer (with dNTPs) comprises 75–150 mM Tris HCl (pH 8.4), 200–500 mM KCl, 5–15 mM MgCl2, 0.5–2 mM dATP, 0.5–2 mM dTTP, 0.5–2 mM dCTP, and 0.5–2 mM dGTP. In this embodiment, it comprises 100 mM Tris HCl (pH 8.4), 250 mM KCl, 7.5 mM MgCl2, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, and 1 mM dGTP.

[0208] b. Taq DNA polymerase, purchased from ShuoShi Biotechnology, catalog number CSP-003-250U;

[0209] c. DMSO was purchased from Merck Biotechnology;

[0210] d. dATP, dTTP, dCTP, dGTP, dUTP, UDG, and Nuclease-free water were purchased from Thermo Fisher Scientific.

[0211] The PCR reaction procedure is as follows:

[0212] Step 1: 95℃ for 5 minutes, 1 cycle;

[0213] Step 2: 95℃ for 30 seconds, 63℃ for 3 minutes, 30 cycles;

[0214] Step 3: 72℃ for 3 minutes, 1 cycle.

[0215] (4) Digestion and terminal repair plus A reaction:

[0216] Mix the two tubes of PCR products from the previous round, and then add the enzymes and reagents as shown in Table 11 below:

[0217] Table 11 Digestion and Terminal Repair Plus A Reaction System

[0218]

[0219] The digestion and final addition of A reaction procedure are as follows:

[0220] Step 1: 37℃ for 15 minutes, 1 cycle;

[0221] Step 2: 80℃ for 20 minutes, 1 cycle;

[0222] Step 3: 65℃ for 15 minutes, 1 cycle;

[0223] (5) Joint connection reaction:

[0224] In the previous round of digestion and final A-addition product, add enzymes and reagents according to the system shown in Table 12:

[0225] Table 12 Joint Connection Reaction System

[0226]

[0227] In the above table:

[0228] a. Ligatin Buffer is a ligase buffer comprising 100–500 mM Tris-HCl, 10–40 mM MgCl2, 1–4 mM DTT, 1–4 mM ATP, and 7.5–30% (v / v) PEG 6000. In this example, it comprises 400 mM Tris-HCl, 40 mM MgCl2, 4 mM DTT, 4 mM ATP, and 30% (v / v) PEG 6000.

[0229] b. T4 DNA Ligase was purchased from Yisheng Biotechnology;

[0230] c. DNA Adapter X for Illumina is a TruSeq DNA UD Indexes (24 indexes, 96 samples) product;

[0231] d. ddH2O is nuclease-free water, purchased from Thermo Fisher Scientific.

[0232] The connector connection reaction procedure is as follows:

[0233] Step 1: 20 ℃ for 15 min, 1 cycle.

[0234] (6) Dual-selection purification of reaction products by linker connection

[0235] A. Equilibrate the DNA purification magnetic beads to room temperature;

[0236] B. Add 50 µL of DNA purification magnetic beads to the library enrichment PCR product from the previous step, mix thoroughly, and let stand for 5 min.

[0237] 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 transfer the supernatant to a new centrifuge tube using a pipette.

[0238] D. Add 40 µL of DNA purification magnetic beads to the supernatant from the previous step, mix thoroughly, and let stand for 5 min.

[0239] 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.

[0240] F. Add 500 µL of freshly prepared 80% (v / v) ethanol solution, let stand for 30 s, and then discard the supernatant;

[0241] G. Repeat the previous step once;

[0242] H. Keep the centrifuge tubes in the magnetic rack at all times, and open the lid to air dry the magnetic beads for 5-10 minutes until there is no ethanol residue.

[0243] 1. Add 22 µL of sterile ultrapure water to elute, gently pipette to mix thoroughly, let stand at room temperature for 5 min, briefly centrifuge the centrifuge tube and place it on a magnetic rack to stand until the solution is clear (about 2 min), carefully transfer 20 µL of supernatant to a new centrifuge tube, being careful not to touch the magnetic beads.

[0244] (7) Document quality control:

[0245] Qseq100 was used to detect the size of the products, and qubit 2.0 was used for library quality control to determine the concentration. The distribution of the library fragments is as follows: Figure 3 As shown, the library fragments are distributed between 200 and 600 bp, with the main peak at 400 to 450 bp. The peak shape is concentrated, which meets the requirements for fragment size for sequencing.

[0246] (8) Sequencing

[0247] The obtained libraries were standardized and mixed in equal amounts. The resulting libraries were then subjected to parallel sequencing using the Illumina NextSeq550Dx sequencing platform and the PE150 sequencing type.

[0248] (9) Data Analysis

[0249] The analysis steps include basic steps such as filtering adapter primer sequences, primer excision, alignment, and output of consistent sequences.

[0250] Results: The RNA extracted from the SARS-CoV-2 pseudovirus was used for library construction three times. The alignment rate was over 99% and the genome coverage was over 99.5%. The full-length viral genome was obtained and correctly genotyped in all cases. The library construction results were good, as shown in Table 13 below.

[0251] Table 13 Results of COVID-19 Database Construction

[0252]

[0253] Conclusion: The method for constructing libraries using PCR long amplicon is simple, can remove aerosol contamination from PCR amplification, and produces libraries of good quality that meet the requirements for instrumentation.

[0254] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can 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 next-generation sequencing library of a long amplicon of PCR, characterized by, The method comprises the following steps: S1, PCR amplification: target sequences are amplified by PCR using primers to obtain PCR long amplicons; S2, digestion and fragmentation of the PCR long amplicons by a mixed enzyme system, and end repair and A addition; the mixed enzyme system comprises Exo I enzyme, UDG enzyme and Taq DNA polymerase; S3, ligation of the A addition product with a sequencing adapter to obtain an adapter-ligated product; S4, purification of the adapter-ligated product: the adapter-ligated product is subjected to a first round of purification treatment; In step S1, when performing PCR amplification, the PCR reaction system comprises primers, dNTPs, a DNA polymerase and a template DNA; the PCR reaction system further comprises UDG enzyme; the amount of the UDG enzyme is 0.1-2 U; Before denaturation, annealing and extension in the PCR amplification reaction program, incubation is first performed at 37°C; In step S2, when constructing the reaction system for digestion, fragmentation and end repair and A addition, the amount of added Exo I enzyme is 5-40 U, the amount of added UDG enzyme is 0.1-2 U; and dATP is supplemented, and the concentration of dATP in the reaction system is 0.1-0.4 mM; The reaction program of step S2 comprises: S21: 37°C for 5-20 min, 1 cycle; S22: 80°C for 10-20 min, 1 cycle; S23: 65°C for 10-30 min, 1 cycle.

2. The method for constructing a next-generation sequencing library according to claim 1, characterized in that, In the PCR reaction system of step S1, 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 amount of the Taq DNA polymerase is 1-5 U; In step S1, the reaction program for PCR amplification comprises: S11: 95°C for 2-5 min, 1 cycle; S12: 95°C for 10-30 s, 55-72°C for 20 s-5 min, 20-35 cycles; S13: 72°C for 3 min, 1 cycle.

3. The method of claim 1, wherein the library is a next generation sequencing library. The reaction system of step S3 comprises a ligase, a sequencing adapter and the reaction product obtained in step S2; the ligase comprises T4 DNA ligase, and the amount of the T4 DNA ligase is 1000-6000 U; And / or, the reaction conditions for adapter ligation in step S3 are 20°C for 10-30 min.

4. The method of claim 3, wherein, The reaction system of step S3 further comprises a ligation reaction buffer, and the ligation reaction buffer contains 100-500 mM Tris-HCl, 10-40 mM MgCl2, 1-4 mM DTT, 1-4 mM ATP and 7.5-30% PEG 6000.

5. The method of claim 1, wherein, The method further comprises the following steps: S5, library enrichment: the purified adapter-ligated product is used as a template for PCR amplification to enrich the library; S6, purification of the library enrichment product: The library enrichment product is subjected to a second round of purification to obtain a second-generation sequencing library.

6. The method of claim 5, wherein, In step S5, the reaction system for library enrichment PCR amplification comprises: a pair of universal library primers, a nucleic acid amplification reaction solution, and the purified adapter ligation product obtained in step S4; the nucleic acid amplification reaction solution comprises dNTPs and a DNA polymerase.

7. Use of the method for constructing a second-generation sequencing library according to any one of claims 1-6 in second-generation sequencing.

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