Construction method of third-generation metagenome sequencing library

Abstract

The invention relates to the technical field of metagenomes and three-generation sequencing, and particularly provides a construction method of a three-generation metagenome sequencing library. According to the method, MDA amplification is introduced into third-generation genome sequencing through T7 incision enzyme treatment, it is guaranteed that the sufficient initial amount of third-generation library building is met, the problem that the total amount of nucleic acid of a metagenome is not suitable for third-generation sequencing is solved, the third-generation library building process is further optimized, and the construction efficiency is improved. The problem that three-generation sequencing cannot be met due to difficult metagenome extraction and poor nucleic acid quality is further solved, the assembly integrity of the metagenome is improved, and deeper function exploration is achieved for diversity research.

Description

Technical Field

[0001] The present invention relates to the technical field of metagenomics and third-generation sequencing, and specifically provides a method for constructing a third-generation metagenomics sequencing library. Background Art

[0002] Microbiome, as an emerging multidisciplinary cross-field, is changing our understanding of life systems at an unprecedented rate. Metagenomic sequencing, as the core tool for microbiome research, is a new microbial research method that directly analyzes the total DNA in the environment or organisms to study microbial diversity, population structure, evolutionary relationships, functional activity, mutual cooperation, and the relationship between microorganisms and the environment. However, the challenge of metagenomic sequencing is to handle complex samples, especially those containing low-abundance microorganisms or high GC content DNA, which will limit the efficiency and accuracy of traditional sequencing methods.

[0003] With the rise of third-generation sequencing technologies, such as PacBio's single-molecule real-time sequencing technology and ONT's nanopore single-molecule technology, not only has the field of sequencing been broadened, but it has also made up for the shortcomings of second-generation sequencing technology in genome assembly and species identification by providing longer read lengths and higher sequencing accuracy. Third-generation sequencing technology has significant advantages for complex ecological environmental studies that require precise genome assembly and in-depth species identification. For example, when studying BVAB1, an uncultured microorganism associated with bacterial vaginosis, long-read third-generation sequencing can reveal the subtle structure of its genome, thereby gaining a deep understanding of the role of the microorganism in the occurrence of the disease and providing new clues for disease prevention and treatment.

[0004] When dealing with metagenomics, traditional library construction methods often lead to difficulties in library construction and poor sequencing data due to factors such as insufficient total DNA nucleic acid and poor quality. These may affect the accurate assessment of species abundance and the complete capture of functional genes, thereby limiting the depth and breadth of metagenomics research. Summary of the invention

[0005] The purpose of the present invention is to develop an optimized third-generation metagenomic sequencing library construction method, which has important scientific significance and practical application value for improving sequencing efficiency and accuracy, reducing noise, and improving species coverage and genome uniformity.

[0006] The present invention aims to provide a method for constructing a metagenomic library, which, combined with the long-read sequencing of the third generation, can efficiently and accurately capture microbial diversity in complex samples, and provide more in-depth molecular information for microbial community research, disease diagnosis, and environmental monitoring. Before constructing the third-generation conventional library, the whole genome amplification (MDA) technology is added to solve the problem of the total amount of nucleic acid in the third-generation metagenomic library construction.

[0007] In a first aspect, the present invention provides a method for constructing a third-generation metagenomic sequencing library, comprising: obtaining a purified product after MDA amplification, magnetic bead purification, T7 endonuclease treatment and a second magnetic bead purification of the DNA of a metagenomic sample; and using the purified product to construct a third-generation metagenomic sequencing library.

[0008] MDA amplification technology is a technology for non-selective amplification of whole genome sequences. Its purpose is to significantly increase the total amount of DNA without sequence bias. This technology uses multiple displacement amplification (MDA) technology to stably amplify genomic DNA. By using random six-base primers to anneal with template DNA at multiple sites, and then using Phi29 DNA polymerase with high amplification efficiency and fidelity to initiate replication at multiple sites of DNA at the same time, synthesize DNA along the DNA template, and replace the complementary chain of the template at the same time. This amplification method can ultimately obtain a large amount of high molecular weight DNA, ensuring the length and coverage of the amplified product.

[0009] Secondly, the Phi29 DNA polymerase used in the MDA amplification technology has a strong template binding ability and can continuously amplify 100Kb DNA templates without dissociating from the template. At the same time, this enzyme has 3' exonuclease activity, which can ensure the high fidelity of amplification. This makes the MDA amplification technology have significant advantages in both the high fidelity and yield of the amplified products.

[0010] In summary, MDA amplification technology has obvious advantages over conventional amplification technology in terms of length and coverage guarantee of amplified products, high fidelity and wide application, and has shown significant advantages in genomic research, especially in application scenarios that require high-quality DNA library construction.

[0011] Although MDA technology has its unique chain displacement amplification mechanism and high-efficiency product quality, MDA amplification is carried out using random hexamer primers, which will form a branched structure during the amplification process. Libraries with branched structures will cause the sequencing enzyme to be unable to synthesize continuously during the PB sequencing process, which will lead to the termination of the sequencing enzyme process, thereby affecting the overall sequencing results; libraries with branched structures will affect the motor protein helical decoupling activity during the ONT sequencing process, thereby affecting the overall sequencing results.

[0012] T7 endonuclease can recognize and cut incompletely paired DNA, cruciform structure DNA, Holliday structure or cross DNA, heteroduplex DNA, and can also cut nicked double-stranded DNA at a slower speed. The present invention uses T7 endonuclease to treat the MDA product, effectively reducing the branching structure in the MDA amplification process, so that the MDA technology can be used in the construction of high-quality DNA libraries.

[0013] The construction method provided by the present invention is applicable to a PB sequencing platform or an ONT sequencing platform.

[0014] More specifically, the construction method provided by the present invention is applicable to sequencing on the PB sequencing platform Revio sequencer and sequencing on the ONT sequencing platform PromethION 48 sequencer.

[0015] In the construction method provided by the present invention, the kit used for MDA amplification is Discover-scSingle Cell WGA Kit from Novezan, catalog number: N603-01.

[0016] In the construction method provided by the present invention, the kit used for magnetic bead purification is Beckman's AgencourtAMPure XP 5mL Kit, catalog number: A63880.

[0017] In the construction method provided by the present invention, the kit used for T7 endonuclease treatment is T7 Endonuclease I from Novezan, catalog number: EN303-01.

[0018] In the construction method provided by the present invention, when the sequencing platform used is a PB sequencing platform, the construction method comprises: S1: Perform MDA amplification on the DNA of the metagenomic sample to obtain the MDA amplification product; S2: treating the MDA amplification product with T7 enzyme; S3: DNA damage repair, end addition of A; S4: Adapter ligation and enzyme treatment to obtain the third-generation library to be sequenced.

[0019] In the construction method provided by the present invention, when the sequencing platform used is a PB sequencing platform, the construction method comprises: S1: Perform MDA amplification on the DNA of the metagenomic sample to obtain the MDA amplification product; S2: treating the MDA amplification product with T7 enzyme; S3: DNA damage repair; S4: Connectors are connected to obtain the third-generation library to be sequenced.

[0020] In a second aspect, the present invention provides an application of the above-mentioned construction method in reducing the workload of metagenome splicing and annotation, sequencing the sequencing library obtained by the construction method, obtaining sequencing data, and performing bioinformatics analysis.

[0021] In a third aspect, the present invention provides an application of the above-mentioned construction method in improving the quality and integrity of metagenomic data, sequencing is performed on the sequencing library obtained by the construction method, sequencing data is obtained, and bioinformatics analysis is performed.

[0022] The present invention fully utilizes the advantages of the long length of third-generation sequencing (Pacbio, ONT), reduces the workload of subsequent genome splicing and annotation, and greatly improves the quality and integrity of the data.

[0023] The beneficial effects of the present invention are: (1) The present invention provides a method for constructing a PB metagenomic library, which increases and purifies DNA samples through MDA and DNA sample pretreatment, thereby solving the problem of difficulty in extracting metagenomics and poor nucleic acid quality that does not meet the requirements of PB sequencing, and improving the assembly integrity of the metagenomics; (2) The present invention provides a method for constructing an ONT metagenomic library, which increases and purifies DNA samples through MDA and DNA sample pretreatment, thereby solving the problem of difficulty in extracting metagenomics and poor nucleic acid quality that does not meet ONT sequencing requirements, and improving the assembly integrity of the metagenomics; (3) The present invention has important scientific significance and practical application value in improving the sequencing efficiency and accuracy of metagenomes, and increasing species coverage and genome uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The present invention provides a flow chart of the construction method.

[0026] Figure 2 This is a bar chart of species composition obtained by second-generation sequencing of the microbial sample of the present invention, and third-generation sequencing of the microbial sample after MDA purification.

[0027] Figure 3 A bar graph of species composition after normal third-generation sequencing and the third-generation sequencing provided by the present invention.

[0028] Figure 4 The species composition histograms of the PB library and the ONT library constructed by the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the raw materials and reagents involved are all commercially available conventional raw materials and reagents, unless otherwise specified; the detection, testing, preparation methods, etc. involved are all conventional.

[0031] In the embodiment of the present invention, the kit used for MDA amplification is Discover-sc Single Cell WGA Kit from Novezan, catalog number: N603-01.

[0032] The kit used for magnetic bead purification is Beckman's Agencourt AMPure XP 5mL Kit, catalog number: A63880.

[0033] The kit used for T7 endonuclease treatment is Novezan's T7 Endonuclease I, catalog number: EN303-01.

[0034] The kit used for PB library construction was Pacbio's SMRTbell Prep Kit 3.0, catalog number: 102-141-700.

[0035] The kit used for AMPure PB magnetic bead screening is Pacbio's AMPure PB Beads, catalog number: 100-265-900.

[0036] The reagents used for DNA repair were produced by NEB, with catalog numbers: M6630S and E7180S.

[0037] The adapter was connected using ONT's ligation sequencing kit V14, catalog number: SQK-LSK114.

[0038] Example 1 Purification of DNA samples for PB sequencing This embodiment provides a third-generation library construction method based on PB sequencing (such as Figure 1 As shown), in this embodiment, the total amount of DNA sample is greater than 10 ng, the electrophoresis gel image has an obvious main band (greater than 23K), and the brightness of the degradation band cannot be higher than the main band.

[0039] The purification method of the DNA sample in this embodiment includes: 1. MDA amplification The basic principle of MDA is to use random hexamer primers and phi29 DNA polymerase to amplify under constant temperature conditions. During the reaction, the primer hexamer first randomly binds to the DNA template, and then the phi29 DNA polymerase starts to replicate at multiple sites of the DNA at the same time. The replicated synthetic DNA replaces the complementary strand of the template, and the replaced complementary strand can become a new template for amplification.

[0040] (1) Prepare Buffer D1 and Stop Solution Mix (the system in the table below is for 12 reactions. Any unused solution in one experiment can be stored at -20°C for no more than 3 months).

[0041] Prepare Buffer D1 with the following components:

[0042] Prepare Stop Solution Mix with the following components:

[0043] (2) Add 2.5 μL of DNA sample (10 ng) into the PCR tube. If the sample volume is less than 2.5 μL, use water or TE to make up to 2.5 μL.

[0044] (3) Add 2.5 μL of Buffer D1, gently tap the tube to mix, and centrifuge briefly.

[0045] (4) Incubate at room temperature for 3 min.

[0046] (5) Add 5 μL Stop Solution Mix, gently tap the tube to mix, and centrifuge briefly. Keep the sample on ice before the next reaction.

[0047] (6) Prepare a reaction mixture with the following components.

[0048] When preparing the reaction mixture, first mix H2O and Discover-sc WGA Master Buffer, then vortex and briefly centrifuge. Add Discover-sc WGA Enzyme Mix and use immediately.

[0049] (7) Immediately add 40 μL of the reaction mixture to the prepared 10 μL DNA sample (1.5), gently tap the tube to mix, and centrifuge briefly.

[0050] (8) Incubate at 30°C for 2 h.

[0051] (9) Incubate at 65°C for 5 min to inactivate Discover-sc WGA Enzyme Mix.

[0052] (10) When using Qubit to measure the concentration, the concentration needs to be greater than 40 ng / μL.

[0053] 2. Magnetic bead purification (1) Take out the XP magnetic beads stored at 4°C in advance, shake and mix, and equilibrate at room temperature for more than 30 min.

[0054] (2) Transfer the product to a 1.5 ml low-absorption centrifuge tube, add 1 volume (about 50 μL) of XP magnetic beads, flick 6-10 times with your fingers to mix, and centrifuge for 2 seconds.

[0055] (3) Let stand at room temperature for 15 minutes.

[0056] (4) After the rest period, remove the LoBind tube and disconnect it for 2 seconds.

[0057] (5) Transfer the LoBind tube to a magnetic rack and let it stand until it becomes clear.

[0058] (6) Aspirate the supernatant and keep it in another labeled 1.5 mL centrifuge tube (if the DNA is not recovered in the end, the supernatant can be re-purified with magnetic beads to recover the DNA). When aspirating the supernatant, be careful not to touch or absorb the magnetic beads.

[0059] (7) Add 200 μL of 70% ethanol, being careful not to wash the magnetic beads. After 30 seconds, aspirate the supernatant, being careful not to touch the magnetic beads during the process.

[0060] (8) Repeat step (7) once, centrifuge the LoBind tube after removing the supernatant for 2 seconds, and place it on a magnetic rack to let it stand until it becomes clear.

[0061] (9) After adding 45 μL Nuclease-Free Water, flick the tube 6-10 times with your finger and briefly rinse for 2 seconds.

[0062] (10) After the standing period, remove the LoBind tube, spin for 2 seconds, place it on a magnetic rack, and let it stand until it becomes clear. Pipette 45 μL of the supernatant and collect it in another labeled 1.5 mL LoBind tube, being careful not to absorb the magnetic beads.

[0063] (11) Take 1 μL of the purified sample for Qubit assay. If the remaining total DNA is greater than 1.5 μg, it is qualified and proceed to the next step. If it is unqualified, take genomic DNA and re-amplify it with MDA.

[0064] (12) The purified DNA can be stored for 1 week.

[0065] 3. T7 endonuclease treatment T7 Endonuclease I (T7 Endo I, T7EI), also known as T7 nuclease I, is a special DNA endonuclease that can recognize and cut non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, and heteroduplex DNA. The cutting site of T7EI is located at the first, second, or third phosphodiester bond at the 5' end of the mismatched base. In addition, T7EI can also cut nicked double-stranded DNA at a very slow rate.

[0066] (1) Add the reagents described in the following table to 44 μL of purified product (for a maximum of 3 ug of purified product)

[0067] (2) React in a metal bath at 37°C for 30 min.

[0068] 4. Magnetic bead purification The magnetic bead purification in this step is the same as that in step 2, except that: Transfer the product in step (2) to a 1.5 mL low-absorption centrifuge tube, add 0.45 times the volume (about 22.5 μL) of XP magnetic beads, flick 6-10 times with your fingers to mix, and centrifuge for 2 s.

[0069] Step (9) After adding 47 μL Nuclease-Free Water, flick the tube 6-10 times with your finger and briefly rinse for 2 seconds.

[0070] After step (10) is completed, remove the LoBind tube, spin for 2 seconds, place it on a magnetic rack, let it stand until it is clear, aspirate 47 μL of the supernatant, and collect it in another labeled 1.5 mL LoBind tube, being careful not to aspirate the magnetic beads.

[0071] Step (11) Take 1 μL of the purified sample for Qubit assay. If the remaining DNA is greater than 1 μg, it is qualified and proceed to the next step. If it is unqualified, take genomic DNA and perform MDA amplification again.

[0072] Example 2 PB library construction The PB library was constructed based on the purified DNA sample in Example 1, and the steps were as follows: 1. DNA repair / addition of A Add the following reagents to the centrifuge tubes:

[0073] (2) Use a wide-mouth pipette tip to gently pipette 15 times to mix the sample and centrifuge for 1 second.

[0074] (3) Incubate at 37°C for 45 min, incubate at 65°C for 5 min, and then return to the ice box.

[0075] 2. Connector connection The reagents used for the adapter linking are as follows:

[0076] Use a wide-mouth pipette tip to gently pipette 15 times to mix the above samples, centrifuge for 1 second, incubate at 20℃ for 45 minutes, then return to the ice box and continue the next step of the reaction.

[0077] 3. SMRTbell Cleanup Beads purification (1) Add 95 μL (1*) of Cleanup Beads to 95 μL of reaction solution 2. The beads need to be equilibrated at room temperature for 30 minutes before use.

[0078] (2) Use a wide-mouth pipette tip to gently pipette 15 times to mix the sample and centrifuge for 1 second.

[0079] (3) Leave at room temperature for 10 minutes and then remove from the heat for 1 second.

[0080] (4) Transfer the centrifuge tube to the magnetic rack until the magnetic beads are completely adsorbed, and transfer the supernatant to a new centrifuge tube to avoid adsorbing the magnetic beads.

[0081] (5) Wash the beads with 1.5 ml of freshly prepared 80% alcohol and discard the alcohol after 30 seconds. Note: Do not remove the tube from the magnetic stand; do not touch the beads.

[0082] (6) Repeat step (5).

[0083] (7) Remove the centrifuge tube from the magnetic rack, spin for 1 second, put it back on the magnetic rack, and discard the alcohol at the bottom of the tube.

[0084] (8) Add 41 μL of Elution Buffer to the tube and mix by gently pipetting 15 times using a wide-mouth pipette tip.

[0085] (9) Dissolve the DNA in 10-15 minutes and centrifuge for 1 second.

[0086] (10) Pipette the supernatant into a new 5 mL low-absorption centrifuge tube. (11) Take 1 μL and dilute 5 times, and use Qubit to measure the concentration. The purified DNA sample can be stored at 4°C for 2 weeks, and can be stored at -20°C for a long time, but avoid repeated freezing and thawing.

[0087] 4. Enzyme treatment The reagents used in the enzyme treatment system are as follows:

[0088] Use a wide-mouth pipette tip to gently pipette 15 times to mix the above samples, centrifuge for 1 second, incubate at 37℃ for 30 minutes, strictly control the time, then put it back in the ice box and continue the next step of the reaction.

[0089] 5. AMPure PB Magnetic Bead Screen The reagents used are as follows:

[0090] (1) Add 185 μL (3.7 times) of diluted AMPure PB beads to the reaction product in the previous step. Note: The beads need to be equilibrated at room temperature for 30 minutes before use.

[0091] (2) Use a wide-mouth pipette tip to gently pipette 15 times to mix the sample and centrifuge for 1 second.

[0092] (3) Leave at room temperature for 15 minutes and then remove from the heat for 1 second.

[0093] (4) Transfer the centrifuge tube to the magnetic rack until the magnetic beads are completely adsorbed, and transfer the supernatant to a new centrifuge tube to avoid adsorbing the magnetic beads.

[0094] (5) Wash the beads with 1.5 ml of freshly prepared 80% alcohol and discard the alcohol after 30 seconds. Note: Do not remove the tube from the magnetic stand; do not touch the beads.

[0095] (6) Repeat the previous step (5).

[0096] (7) Remove the centrifuge tube from the magnetic rack, spin for 1 second, put it back on the magnetic rack, and discard the alcohol at the bottom of the tube.

[0097] (8) Add 16 μL of 1X Elution Buffer to the tube and mix by gently pipetting 15 times using a wide-mouth pipette tip.

[0098] (9) Dissolve the DNA at 37°C for 15 minutes and centrifuge for 1 second.

[0099] (10) Pipette 16 μL of supernatant into a new 1.5 mL low-absorption centrifuge tube. (11) Take 1 μL and dilute it 5 times. Use Qubit to measure the concentration and Aglient 5300 to detect the fragment length.

[0100] (12) The qualified libraries are then put on the machine for subsequent sequencing.

[0101] 6. PB library combined with on-machine sequencing (1) SMRT Link, click to enter Sample Setup to set library information.

[0102] (2) Generate a binding process based on the set parameters, and combine the sequencing primers and sequencing enzymes in the library according to the process.

[0103] (3) Use Revio sequencer to sequence the binding products.

[0104] (4) Statistics and quality control of sequencing results.

[0105] The species composition bar graph obtained by the second generation sequencing of the microbial samples used in this example ( Figure 2 ), and the species composition bar graph obtained from the third-generation sequencing data obtained in this example ( Figure 2 denoted as MDA in Figure 2 .

[0106] Depend on Figure 2 It can be seen that the species composition ratios of the 10 species in the third-generation sequencing data obtained by the construction method provided by the present invention are basically the same as those in the second-generation sequencing data, which proves that the results of this process have no species bias compared with the second-generation data.

[0107] Normal third-generation sequencing ( Figure 3 The process marked in the figure is normal, and the standard operation process of Pacbio manufacturer is adopted, Preparing whole genome and metagenome libraries using SMRTbell® prep kit3.0) and the third-generation sequencing of the present invention ( Figure 3 The species composition bar graph obtained by Figure 3 , the sequencing data results are as follows:

[0108] Depend on Figure 3 It can be seen that compared with ordinary third-generation sequencing data, the species composition ratios of 10 species in the third-generation sequencing data obtained using the construction method provided by the present invention are basically the same, proving that the results of this process have no species bias compared to normal third-generation data.

[0109] Example 3 Purification of DNA samples based on ONT sequencing The DNA sample used in this example has a total amount greater than 10 ng, an obvious main band (greater than 23K) in the electrophoresis gel image, and the brightness of the degradation band cannot be higher than that of the main band.

[0110] The purification method of the DNA sample in this embodiment is as follows: 1. MDA amplification, 2. magnetic bead purification, and 3. T7 endonuclease treatment are all the same as 1. MDA amplification, 2. magnetic bead purification, and 3. T7 endonuclease treatment in Example 1.

[0111] The difference between step 4, magnetic bead purification in this embodiment and the magnetic bead purification in step 2 in this embodiment is that: In step (2), transfer the product to a 1.5 ml low-absorption centrifuge tube, add 0.45 times the volume (about 22.5 μL) of XP magnetic beads, flick the tube 6-10 times with your fingers to mix, and centrifuge for 2 seconds.

[0112] Step (9) After adding 49 μL Nuclease-Free Water, flick the tube 6-10 times with your finger and briefly rinse for 2 seconds.

[0113] After step (10) is completed, remove the LoBind tube, spin for 2 seconds, place it on a magnetic rack, let it stand until it is clear, aspirate 49 μL of the supernatant, and collect it in another labeled 1.5 mL LoBind tube, being careful not to aspirate the magnetic beads.

[0114] Step (11) Take 1 μL of the purified sample for Qubit detection. If the remaining total DNA is greater than 1 μg, it is qualified and proceed to the next step. If it is unqualified, take genomic DNA and re-amplify it with MDA.

[0115] Example 4 ONT library construction This example provides a method for constructing an ONT library using the purified DNA from Example 3, the steps being as follows: 1. DNA repair (1) Prepare a 0.2 mL LoBind tube and prepare the reaction solution according to the following ratio:

[0116] (2) Mix by tapping and then briefly stirring for 2 seconds.

[0117] (3) Use a PCR instrument, cover the PCR instrument with a hot cover, and run the program in the following table. After the PCR instrument's hot cover has finished heating up (75°C), the temperature of the PCR instrument drops to 20°C, open the hot cover, and transfer the sample tube to the PCR instrument for reaction.

[0118] 2. Magnetic bead purification (1) Take out the XP magnetic beads stored at 4°C in advance, shake and mix, and equilibrate at room temperature for more than 30 min.

[0119] (2) Add 60 μL (1 volume) of XP magnetic beads to the sample, flick the sample 6-10 times to mix, centrifuge for 2 seconds, and then flick the sample 6-10 times to mix again without further centrifugation.

[0120] (3) Let stand at room temperature for 5 min.

[0121] (4) After the rest period, remove the LoBind tube and disconnect it for 2 seconds.

[0122] (5) Transfer the LoBind tube to a magnetic rack, let it stand until it becomes clear, and discard the supernatant.

[0123] (6) Add 200 μL of 70% ethanol to the suspension, being careful not to wash the magnetic beads. After 30 seconds, aspirate the supernatant, and do not touch the magnetic beads during the process.

[0124] (7) Repeat step (6) once, centrifuge the LoBind tube after removing the supernatant for 2 seconds, and place it on a magnetic stand until it becomes clear. Remove the remaining ethanol with a 10 μL pipette, then open the lid and let it dry until the magnetic beads lose their luster.

[0125] (8) After adding 31 μL Nuclease-Free Water, flick the tube 6-10 times with your finger, pause for 2 seconds, flick the tube 6-10 times again with your finger, and let it stand at room temperature for 3 min.

[0126] (9) After the standing period, remove the LoBind tube, spin for 2 seconds, place it on a magnetic rack, and let it stand until it becomes clear. Pipette 30 μL of the supernatant and collect it in another labeled 1.5 mL LoBind tube. Be careful not to absorb the magnetic beads.

[0127] (10) Take 1 μL of the purified sample for Qubit detection (the remaining total DNA amount is greater than 1 μg), and the DNA sample is immediately subjected to the next step of reaction. It can be temporarily stored at 4°C for about 1 hour, and is strictly prohibited from being stored at -20°C.

[0128] 3. Connector connection Take a LoBind tube, place it on ice, and set up the reaction system as shown in the table below:

[0129] Mix by flicking, centrifuge briefly, and incubate at room temperature for 1 hour.

[0130] 4. Magnetic bead purification (1) Add 20 μL (0.4X) of resuspended AMPure XP beads to the reaction and flick the tube to mix. Incubate at room temperature for 5 minutes.

[0131] (2) Place the tube on a magnetic rack, wait for the sample and magnetic beads to be adsorbed to the magnet, and remove the supernatant.

[0132] (3) Add 125 μL of long fragment buffer (LFB) to wash the beads. Flick the beads to resuspend them, then place the tube back on the magnetic rack and allow the beads to adsorb. Transfer the supernatant to a 1.5 mL LoBind tube for temporary storage.

[0133] (4) Repeat the previous step.

[0134] (5) After a short 2-second spin, place the tube back on the magnet. Use a 10 μL pipette to remove the remaining supernatant. Allow the magnetic beads to dry until they lose their luster.

[0135] (6) Remove the centrifuge tube from the magnetic rack, add 15 μL of EB, flick gently to resuspend, and incubate at 37°C for 10 min, flicking gently every 2 min to mix.

[0136] (7) After the incubation, remove the LoBind tube, spin for 2 seconds, place it on a magnetic rack, and let it stand until it becomes clear. Pipette 15 μL of the supernatant and transfer it to a new 1.5 mL LoBind tube. Gently flick to mix.

[0137] (8) Take 1 μL of the purified sample for Qubit assay.

[0138] (9) The qualified libraries are then put on the machine for subsequent sequencing.

[0139] 5. Sequencing of ONT library (1) Prepare the Library Mix and slowly inject it into the flow cell through the sample injection hole.

[0140] (2) Run the ONT program to sequence the library.

[0141] (3) Statistics and quality control of sequencing results.

[0142] The species composition histograms of the PB and ONT libraries are shown in Figure 4 , the comparison of ONT library and PB library assembly results is as follows:

[0143] Depend on Figure 4The results show that the standard DNA samples amplified and purified by MDA provided by the present invention were subjected to third-generation sequencing. The PB or ONT platform was basically the same in terms of 10 species composition. Combined with the assembly results, it was proved that the present invention can obtain good quality sequencing data in all three generations of sequencing.

[0144] The present invention uses the MDA amplification process to build a third-generation sequencing library, providing a new idea for the construction of a third-generation metagenomic library. It solves the problem that the metagenomics cannot be applied to the third-generation sequencing due to the difficulty in extraction, significantly improves the efficiency and quality of sequencing, and provides a strong technical support for metagenomic research. This method is expected to play an important role in the fields of microbiome, medical research, and environmental monitoring in the future.

[0145] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a third-generation metagenomic sequencing library, characterized in that: include: The DNA of the metagenomic samples was amplified by MDA, purified by magnetic beads, treated with T7 endonuclease, and purified by magnetic beads for the second time to obtain the purified product; The purified product was used to construct a third-generation metagenomic sequencing library.

2. The construction method according to claim 1, characterized in that: The construction method is applicable to a PB sequencing platform or an ONT sequencing platform.

3. The construction method according to claim 1, characterized in that: The kit used for MDA amplification was Discover-sc Single Cell WGA Kit from Novezan.

4. The construction method according to claim 1, characterized in that: The kit used for magnetic bead purification was Beckman's Agencourt AMPure XP 5mL Kit.

5. The construction method according to claim 1, characterized in that: The kit used for T7 endonuclease treatment was T7 Endonuclease I from Novizan.

6. The construction method according to any one of claims 1 to 5, characterized in that: When the sequencing platform used is a PB sequencing platform, the construction method includes: S1: Perform MDA amplification on the DNA of the metagenomic sample to obtain the MDA amplification product; S2: treating the MDA amplification product with T7 enzyme; S3: DNA damage repair, end addition of A; S4: Adapter ligation and enzyme treatment to obtain the third-generation library to be sequenced.

7. The construction method according to any one of claims 1 to 5, characterized in that: When the sequencing platform used is a PB sequencing platform, the construction method includes: S1: Perform MDA amplification on the DNA of the metagenomic sample to obtain the MDA amplification product; S2: treating the MDA amplification product with T7 enzyme; S3: DNA damage repair; S4: Connectors are connected to obtain the third-generation library to be sequenced.

8. Application of the construction method according to any one of claims 1 to 7 in reducing the workload of metagenome splicing and annotation, characterized in that: The sequencing library obtained by the construction method is sequenced to obtain sequencing data, and bioinformatics analysis is performed.

9. Application of the construction method according to any one of claims 1 to 7 in improving the quality and integrity of metagenomic data, characterized in that: The sequencing library obtained by the construction method is sequenced to obtain sequencing data, and bioinformatics analysis is performed.

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