Full-length 16s rRNA high-throughput sequencing library construction method based on next-generation sequencing technology and reagent thereof

Abstract

The invention provides a construction method of a full-length 16s rRNA high-throughput sequencing library based on a next-generation sequencing technology and a reagent thereof, and belongs to the technical field of sequencing. The invention provides a quadruple PCR (Polymerase Chain Reaction) amplification primer, which comprises a primer pair for amplifying a 16s rRNA (Ribonucleic Acid) section, and the specific nucleotide sequences of the primer pair are as shown in SEQ ID NO: 1-SEQ ID NO: 8. The invention further provides a full-length 16s rRNA high-throughput sequencing library construction method based on the next-generation sequencing technology, the full-length 16s rRNA sequence is subjected to quadruple PCR amplification by adopting the quadruple PCR amplification primer, a next-generation sequencer is used for sequencing, the one-time sequencing success rate and the seed / strain level annotation accuracy rate are higher than 95.04%, and the purpose of low-cost and high-throughput sequencing is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of sequencing, and in particular relates to a method for constructing a full-length 16S rRNA high-throughput sequencing library based on the second-generation sequencing technology and a reagent thereof. Background Art

[0002] 16s rRNA is the most commonly used molecular biological method for identifying microbial species. The advantage of using 16s rRNA as a microbial identification marker is that in the sequence of about 1500bp in length, there are both highly conserved sequences, reflecting its stability and extensiveness in microbial evolution, and variable regions, which facilitates the distinction of subtle evolutionary relationships between different microorganisms. By comparing the full-length 16s rRNA sequence, microorganisms can be accurately annotated to the strain (type) level. Therefore, 16s rRNA has become one of the gold standards for microbial species identification.

[0003] Traditional 16s rRNA determination relies on Sanger sequencing, which is mature and reliable, but has low efficiency. Pyrosequencing can also be used to determine the full-length 16s rRNA sequence. It has a high sequencing throughput, but a high base misreading rate (1% to 5%), which undoubtedly increases the risk of microbial annotation errors. The same problem also exists in third-generation sequencers. The cost of these methods is relatively high. With the development of next-generation (high-throughput) sequencing, especially the rapid expansion of microbiome research, amplicon sequencing technology focusing on variable region 16s sequences has developed rapidly. At present, the more popular 16s rRNA amplicon sequencing mainly includes V1-V2, V3-V4, V4, V4-V5, V6-V7 and V7-V9 regions. The species annotation capabilities vary, and there is a certain degree of distortion for different microbial classifications, but the overall annotation capability is much lower than the effect of the 16s rRNA full-length sequence. Taking the widely used V3-V4 amplicon as an example, usually, no more than 95% of the sequences are annotated to the family level, no more than 90% of the sequences can be annotated to the genus level, no more than 80% of the sequences can be annotated to the species level, and no more than 50% are annotated to the strain (type) level. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a full-length 16s rRNA high-throughput sequencing library construction method and reagents based on the second-generation sequencing technology, which have high identification accuracy and injection accuracy.

[0005] The present invention provides a quadruple PCR amplification primer, comprising a first primer pair for amplifying a 16s rRNA V1-V3 segment, a second primer pair for amplifying a 16s rRNA V3-V5 segment, a third primer pair for amplifying a 16s rRNA V5-V7 segment, and a fourth primer pair for amplifying a 16s rRNA V7-V9 segment;

[0006] The first primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 2;

[0007] The second primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:4;

[0008] The third primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:6;

[0009] The fourth primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:8.

[0010] The present invention provides a reagent for constructing a full-length 16S rRNA high-throughput sequencing library based on the second-generation sequencing technology, including primers for library construction;

[0011] The primer for library construction is formed by sequentially connecting a linker, a custom index and one of the four-plex PCR amplification primers in claim 1.

[0012] Preferably, the linker comprises a linker 1 having a nucleotide sequence as shown in SEQ ID NO:9 and a linker 2 having a nucleotide sequence as shown in SEQ ID NO:10;

[0013] The connector 1 is used to connect the forward primer in the quadruple PCR amplification primer;

[0014] The connector 2 is used to connect the reverse primer in the quadruple PCR amplification primer.

[0015] Preferably, the custom index is an oligonucleotide sequence formed by 2 to 4 random nucleotides in length.

[0016] Preferably, it comprises an amplification reaction premix and a high-fidelity DNA polymerase.

[0017] The present invention provides application of the reagent in constructing a high-throughput sequencing library or high-throughput second-generation sequencing.

[0018] The present invention adopts a method for constructing a full-length 16S rRNA high-throughput sequencing library based on the second-generation sequencing technology, comprising the following steps:

[0019] Using the DNA of the samples to be sequenced as templates, respectively, and using the quadruple PCR amplification primers to perform quadruple PCR amplification to obtain quadruple PCR amplification products;

[0020] The quadruple PCR amplification products are mixed, and the obtained PCR product mixture is used as a template to perform Index PCR amplification using index primers adapted to the Illumina platform to obtain Index PCR amplification products;

[0021] The Index PCR amplification products are mixed to obtain a high-throughput sequencing library.

[0022] Preferably, the reaction system of the quadruple PCR amplification is 25 μL, specifically including 12.5 μL of 2×PCR mix, 0.5 μL of high-fidelity DNA polymerase, 1 μL of each forward primer and reverse primer, and 4 μL of ddH2O.

[0023] Preferably, the reaction procedure of the quadruple PCR amplification is 95°C for 3 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, 25 cycles; 72°C for 5 min.

[0024] The present invention provides a quadruple PCR amplification primer, including a first primer pair for amplifying a 16s rRNA V1-V3 segment, a second primer pair for amplifying a 16s rRNA V3-V5 segment, a third primer pair for amplifying a 16s rRNA V5-V7 segment, and a fourth primer pair for amplifying a 16s rRNA V7-V9 segment, and the specific nucleotide sequence is shown in SEQ ID NO: 1 to SEQ ID NO: 8. The present invention comprehensively considers the distribution of variable regions, the limitations of sequencing equipment, system complexity, and primer reliability. Based on the multiple PCR amplification technology, 4 pairs of PCR primers capable of amplifying different overlapping segments in 16s rRNA are designed to obtain 4 PCR products with overlapping ends, and 4 PCR amplification fragments are continuously assembled, which can realize the sequencing of the full-length 16s rRNA sequence, which greatly improves the accuracy of the sequencing results, and also improves the accuracy of the annotation results. In the embodiment of the present invention, the sequencing library is constructed using the quadruple PCR amplification primer provided by the present invention, and the sequencing result accuracy of the second-generation sequencing is 95.04%, and the annotation accuracy at the strain level is 95.04% on average.

[0025] The present invention also provides a method for constructing a full-length 16s rRNA high-throughput sequencing library based on the second-generation sequencing technology, wherein the DNA of the sample to be sequenced is used as a template, and the quadruple PCR amplification primer is used to perform quadruple PCR amplification, and the obtained quadruple PCR amplification products are mixed, and the obtained PCR product mixture is used as a template, and the index primer adapted to the Illumina platform is used to perform Index PCR amplification to obtain Index PCR amplification products; the Index PCR amplification products are mixed to obtain a high-throughput sequencing library. The sequencing library constructed by the method provided by the present invention makes the sequencing accuracy and annotation accuracy of the second-generation sequencing higher than 95%, and the sequencing accuracy is higher than that of the Sanger sequencing method (90%). At the same time, from the perspective of the measurement cost, the method of the present invention considers that the cost of determining the full-length sequence of 16s rRNA of a single sample is less than 5 yuan, which is much lower than the cost of the Sanger sequencing method (unit price 25 to 30 yuan). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of multiplex PCR of full-length 16s rRNA sequences suitable for second-generation sequencers. DETAILED DESCRIPTION

[0027] The present invention provides a quadruple PCR amplification primer, comprising a first primer pair for amplifying a 16s rRNA V1-V3 segment, a second primer pair for amplifying a 16s rRNA V3-V5 segment, a third primer pair for amplifying a 16s rRNA V5-V7 segment, and a fourth primer pair for amplifying a 16s rRNA V7-V9 segment. The specific nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:8, and the corresponding relationship is shown in Table 1.

[0028] The present invention provides a reagent for constructing a full-length 16S rRNA high-throughput sequencing library based on the second-generation sequencing technology, comprising primers for library construction; the primers for library construction are formed by sequentially connecting a linker, a custom index and one of the quadruple PCR amplification primers described in claim 1.

[0029] In the present invention, the connector preferably includes a connector 1 having a nucleotide sequence as shown in SEQ ID NO:9 and a connector 2 as shown in SEQ ID NO:10. The connector 1 is used to connect the forward primer in the quadruple PCR amplification primer; the connector 2 is used to connect the reverse primer in the quadruple PCR amplification primer. The custom index is preferably an oligonucleotide sequence formed by 2 to 4 random nucleotides in length. In an embodiment of the present invention, the custom index is an oligonucleotide sequence (NN, where N is an arbitrary nucleotide) formed by a random nucleotide sequence of 2 lengths. The custom index is used to distinguish different samples to be tested.

[0030] In the present invention, the reagent preferably includes an amplification reaction premix and a high-fidelity DNA polymerase. The amplification reaction premix and the high-fidelity DNA polymerase are used for quadruple PCR amplification. The present invention has no special restrictions on the sources of the amplification reaction premix and the high-fidelity DNA polymerase, and the sources of the amplification reaction premix and the high-fidelity DNA polymerase known in the art can be used. In an embodiment of the present invention, the amplification reaction premix (containing high-fidelity DNA polymerase) was purchased from Beijing Quanshijin, and the item number is AP221-11.

[0031] In the present invention, the reagent preferably also includes sorting magnetic beads and / or washing solution. The source of the sorting magnetic beads is Hieff DNA Selection Beads DNA selection magnetic beads are used to select DNA fragments of 400-650bp. The washing solution is an ethanol aqueous solution with a volume percentage of 80%.

[0032] The present invention provides application of the reagent in constructing a high-throughput sequencing library or high-throughput second-generation sequencing.

[0033] The present invention adopts a method for constructing a full-length 16S rRNA high-throughput sequencing library based on the second-generation sequencing technology, comprising the following steps:

[0034] Using the DNA of the samples to be sequenced as templates, respectively, and using the quadruple PCR amplification primers to perform quadruple PCR amplification to obtain quadruple PCR amplification products;

[0035] The quadruple PCR amplification products are mixed, and the obtained PCR product mixture is used as a template to perform Index PCR amplification using index primers adapted to the Illumina platform to obtain Index PCR amplification products;

[0036] The Index PCR amplification products are mixed to obtain a high-throughput sequencing library.

[0037] The present invention uses the DNA of the sample to be sequenced as a template, and uses the quadruple PCR amplification primers to perform quadruple PCR amplification to obtain quadruple PCR amplification products.

[0038] In the present invention, the DNA extraction method of the sample to be sequenced preferably includes a kit method, a manual extraction method or extraction using an automatic nucleic acid extractor. The kit method is preferably performed using a Tiangen bacterial DNA extraction kit. The present invention has no special significance for the manual extraction method, and the bacterial DNA extraction can be completed using a manual extraction method well known in the art.

[0039] In the present invention, the reaction system of the quadruple PCR amplification is preferably 25 μL, specifically including 2×PCR mix 12.5 μL, high-fidelity DNA polymerase 0.5 μL, each forward primer and reverse primer 1 μL, ddH2O 4 μL. The reaction procedure of the quadruple PCR amplification is preferably 95°C 3min; 95°C 30sec, 55°C 30sec, 72°C 30sec, 25 cycles; 72°C 5min.

[0040] In the present invention, the quadruple PCR amplification product is preferably purified before Index PCR. The purification method is preferably performed using sorting magnetic beads. The sorting magnetic beads can separate DNA fragments of a certain length by adsorbing DNA fragments of a certain length. DNA Selection Beads When DNA sorting magnetic beads are used for purification, it is preferred to mix the quadruple PCR amplification product with sorting magnetic beads (1:0.6, volume ratio), incubate at room temperature, centrifuge, let stand under the action of magnetic field force until the solution is clear, aspirate the supernatant and transfer it to a new tube, add sorting magnetic beads (1:0.2, volume ratio), incubate at room temperature again, let stand under the action of magnetic field force until the solution is clear, wash the sorting magnetic beads under the action of magnetic field force, and after drying, dissolve the DNA fragments with ddH2O, incubate briefly, remove the sorting magnetic beads, and obtain sorted quadruple PCR amplification products. The room temperature incubation time is preferably 4 to 6 minutes, and can be 5 minutes. The magnetic field force refers to the magnetic force provided by the magnetic frame. The short incubation time is selected to be 25 to 35 seconds, which can be 30 seconds. The purpose of the drying is to remove the residual ethanol on the magnetic beads.

[0041] After obtaining the quadruple PCR amplification products, the present invention mixes the quadruple PCR amplification products, uses the obtained PCR product mixture as a template, and performs Index PCR amplification using index primers adapted to the Illumina platform to obtain Index PCR amplification products.

[0042] In the present invention, the index primer adapted to the Illumina platform is preferably Illumina The XT reagent set includes three 12×8 indexes: SetA, SetB, and SetC. The index PCR amplification was performed according to the Illumina manual.

[0043] After obtaining the Index PCR amplification products, the present invention mixes the Index PCR amplification products to obtain a high-throughput sequencing library.

[0044] In the present invention, the high-throughput sequencing library is denatured and sequenced on the machine. The sequencing reagent is MiSeqReagent Kit v3. The second-generation sequencing platform used for sequencing on the machine is preferably a short sequence determination platform within 600 cycles. After sequencing, the sequencing data is preferably processed. The data processing includes FASTQ splitting and quality control, sequence assembly and 16s rRNA sequence annotation. The FASTQ splitting tool is preferably Illumina CASAVA. The quality control preferably uses the Trimmomatics tool to filter the FASTQ file according to the default parameters. The sequence assembly preferably uses Bwa to assemble the two sequence files R1 and R2 belonging to the same sample, and derives the assembly results. The 16s rRNA sequence annotation is preferably compared one by one with the Diamond tool RDP database (Ribosomal Database Project Ribosomal Database Project, https: / / www.glbrc.org / data-and-tools / glbrc-data-sets / ribosomal-database-project) for species annotation.

[0045] The following is a detailed description of a method for constructing a full-length 16s rRNA high-throughput sequencing library based on the second-generation sequencing technology and reagents provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] When designing 16S rRNA multiplex PCR primers, consider the following design principles:

[0048] Principle 1: The sequence of 16s rRNA contains the conserved region (C1-C10) and the highly variable region (V1-V9). In order to ensure amplification efficiency and high coverage, primers should be designed to the conserved region.

[0049] Principle 2: The read length of current mainstream high-throughput sequencing is 75-600bp. If paired-end sequencing is used, the read length is up to 300bp. Therefore, each PCR should cover less than 600bp to meet the requirements of double-end sequence splicing.

[0050] Principle 3: Multiple PCRs should have appropriate overlap to meet the splicing requirements. The longer the overlapping region, the higher the proportion of high-quality sequence regions.

[0051] Principle 4: Multiplex PCR systems between universal primer groups widely used in academia should be used whenever possible. They should be selected from a large number of universal primers that have been used in the scientific research field for a long time and are widely recognized for their specificity and versatility.

[0052] According to principles 1 to 3, the total number of PCR reactions must not be less than 3, because 2 reactions cannot cover the full-length sequence; when the total number of reactions is 3, it is impossible to obtain primers that are exactly located in the conserved region, so the efficient and accurate amplification of the sequence cannot be guaranteed; and when 4 groups of PCR reactions are designed to cover V1-V2-V3, V3-V4-V5, V5-V6-V7 and V7-V8-V9 respectively, it can be ensured that all universal primers are located in the conserved region, and the lengths of the products obtained from different reactions are moderate, and there is an overlap of no less than 100 bp between the products obtained from different reactions. The universal primers used in this embodiment are shown in Table 1.

[0053] Table 1 Multiplex PCR primers for 16S rRNA full-length sequences

[0054]

[0055]

[0056] Variable regions, primer positions, and amplicon positions are shown in Figure 1 .

[0057] The above primers should be compatible with the adapter sequence of the sequencing platform. This embodiment takes the Illumina platform Truseq scheme as an example. In the primers for the first round of PCR, the forward primer adds an adapter: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 9), and the reverse primer should add 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 10). Of course, in order to achieve the sample volume on the machine, several custom indexes can be added after the adapter primer and before the specific primer. For example, in this embodiment, an "NN" 2-base index is added, which can be used to distinguish 16 different samples. After the primers are synthesized, they are diluted to 10 μM for standby use. The custom index method is the same for other library construction methods based on adapter connection.

[0058] Example 2

[0059] DNA extraction

[0060] 4217 strains of purified bacterial cultures with known species taxonomic relationships were obtained, and 4217 strains of cultures were extracted using a manual bacterial DNA extraction method, including the following steps:

[0061] 1. Take 5 ml of bacterial solution, centrifuge and remove the supernatant, add 50 ml of ddH2O to suspend, boil in boiling water bath for 20 min, freeze in liquid nitrogen, and repeat twice.

[0062] 2. Preheat the cell extract (100mmol / L Tris-HCl, pH 8.0, 5mmo / L EDTA, 500mmol / L NaCl, 1.25% SDS, 1% β-mercaptoethanol) at 65℃;

[0063] 3. Add 500 μL of cell extract, invert, incubate at 65°C for 0.5 h, invert every 10 min.

[0064] 4. Add 150 μL of 5 mol / L potassium acetate (KAC), invert, place on ice for 20 min, and centrifuge at 10,000 rpm for 10 min. Take the supernatant to another EP, add a mixture of phenol, chloroform and isoamyl alcohol (volume ratio of 1:1:1), and centrifuge at 12,000 rpm for 5 min.

[0065] 5. Take the supernatant to another EP tube, add a mixture of phenol, chloroform and isoamyl alcohol (volume ratio of 1:1:1), and centrifuge at 12000 rpm for 5 minutes.

[0066] 6. Add an equal volume of isopropanol, mix gently, and place at -20℃ for 1h.

[0067] 7. Centrifuge at 12000rpm for 3min, discard the supernatant, add 1mL 70% ethanol aqueous solution, centrifuge at 12000rpm for 2min, dry naturally, and dissolve in 20μL ddH2O.

[0068] Example 3

[0069] Multiplex PCR

[0070] The full-length 16s rRNA sequence of the culture described in Example 1 was amplified using a 96-well PCR plate, and the following reaction system was added: 12.5 μL of 2×PCR mix, 0.5 μL of high-fidelity DNA polymerase, 1 μL of each primer, and 4 μL of ddH2O. The amplification conditions were: 95°C, 3 min; 95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec, 25 cycles; 72°C, 5 min.

[0071] After the amplification is completed, every 16 samples (belonging to the same set of custom Index solutions) are mixed and tested by 1.2% agarose gel electrophoresis. There should be two obvious bands at 550bp and 450bp (depending on the electrophoresis conditions, 4 target fragments can be observed under the optimal conditions). After the inspection is correct, enter the subsequent operation process. The merging operation helps to greatly save the reagent consumption in the subsequent operations and significantly saves time. In this embodiment, the number of mixed samples after merging is 264.

[0072] Example 4

[0073] PCR product purification

[0074] PCR products were purified using magnetic beads. Since the properties of magnetic beads from different suppliers were slightly different, the conditions were first optimized to obtain the correct target length range of DNA. DNA Selection Beads DNA sorting magnetic beads are taken as an example, and the purification process is as follows:

[0075] Mix the PCR product with 0.6 times the volume of sorting magnetic beads, incubate at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand, let stand for 5 minutes until the solution is clear, aspirate the supernatant (avoid aspirating the magnetic beads) and transfer to a new tube, add 0.2 times the volume of sorting magnetic beads, mix thoroughly, incubate at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand, let stand for 5 minutes until the solution is clear. Keep on the magnetic stand and wash twice. The washing method is: add 200μL of freshly prepared 80% ethanol aqueous solution to rinse the magnetic beads, incubate at room temperature for 30s, and carefully remove the supernatant. Open the lid and dry on the magnetic stand for 5 minutes. Remove the PCR tube from the magnetic stand, add 20μL ddH2O, vortex and incubate at room temperature for 5 minutes. After a brief centrifugation, place on a magnetic stand, let stand for 5 minutes, and then take the supernatant, which is the sorted DNA.

[0076] Example 5

[0077] High-throughput sequencing

[0078] Since Illumina The three 12×8Index of SetA, SetB, and SetC of the XT reagent set provide a total of 288 Index solutions, which can meet the sequencing requirements of 264 samples in Example 1. The second round of PCR (Index PCR) and PCR product purification were continued according to the Illumina manual, and the DNA was quantified using the Invitrogen Qubit fluorometer. Library mixing, denaturation, and sequencing were performed according to the manual, and the reagent was MiSeq Reagent Kit v3.

[0079] Example 6

[0080] Data processing

[0081] FASTQ splitting and quality control: Illumina CASAVA (or other similar sequence splitting tools) was used to split each FASTQ file into 16 samples based on the first two bases (i.e., the custom Index sequence described in Example 1). Trimmomatics tools were used to filter the FASTQ files.

[0082] Sequence assembly: Use BWA to assemble the two sequence files R1 and R2 belonging to the same sample, and export the assembly results. First, create a reference sequence file, here use the 16s rRNA gene sequence (SEQ ID NO: 11) of Escherichia coli MPEC EX-33 (Genbak accession number: AB045730). Use bwa index reference.fa to index the sequence, use bwa mem reference.fa Sample_R1.fastq Sample_R2.fastq|samtoolsview-S-bF 4->sample.bam to obtain the assembled Bam file, and use samtools faidx sample.bam>sample.fasta to obtain the fasta format sequence file of the sample. 16s rRNA sequence annotation: The obtained sequence is compared with RDP one by one using the Diamond tool for species annotation.

[0083] The statistical results of sequence processing are shown in Table 2.

[0084] Table 2 Statistical results of sequence processing

[0085]

[0086] The statistical results of the annotation accuracy of the sequences sequenced by the method of this embodiment are shown in Table 3.

[0087] Table 3 Statistical results of sequence annotation accuracy

[0088]

[0089]

[0090] Among them, the similarity is the highest similarity of the obtained sequence in the search and comparison of the NT database in GenBank, which indirectly reflects the quality of the sequencing base. If the value is less than 90%, it means that the quality of this sequence is very poor and can hardly be used for species annotation. A total of 14 strains were inaccurately annotated at the genus level; a total of 79 strains were inaccurately annotated at the species / strain level; the accuracy rates at the genus and species / strain levels were 97.98% and 95.04%, respectively. If a single determination, that is, accurate annotation to the species / strain level, is considered a successful sequencing, the one-time success rate of the present invention is 95.04%. The one-time success rate of the Sanger sequencing method is 90%, which is caused by excessive low-quality bases, incorrect interpretation of consecutive identical bases, splicing failures, etc., which lead to about 10% of the one-time sequencing failures, and this part must be resequenced. It can be seen that the present invention can save time, manpower, and material costs through a higher one-time sequencing success rate. In terms of cost comparison, the cost of determining the full-length sequence of 16s rRNA of a single sample is less than 5 yuan, which is much lower than the Sanger sequencing method.

[0091] Comparative Example 1

[0092] Using the same sequencing kit, 192 bacterial strains were selected and high-throughput sequencing of 16s sequences in the V3V4 region was performed according to the Illumina official website manual. Based on the above full-length 16s sequence identification results, the V3V4 region annotation results were statistically analyzed, and the annotation accuracy rates at the genus, species, and strain levels are shown in Table 4:

[0093] Table 4 Annotation accuracy of high-throughput sequencing of V3V4 region at different classification levels

[0094] Classification level Accuracy (%) Genus 90.6 kind 52.1 strain 34.9

[0095] It can be seen that compared with high-throughput sequencing of partial 16s sequences such as V3V4, under the same PE300 sequencing strategy, 16s full-length high-throughput sequencing relying on quadruple PCR can obtain higher genus, species, and strain annotation accuracy. It can be seen that under the read length limit of the second-generation high-throughput sequencer (<300bp), the library constructed by multiplex PCR amplification of 16s has great advantages in annotation accuracy and cost.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quadruple PCR amplification primer, characterized in that: The method comprises a first primer pair for amplifying the 16s rRNA V1-V3 segment, a second primer pair for amplifying the 16s rRNA V3-V5 segment, a third primer pair for amplifying the 16s rRNA V5-V7 segment, and a fourth primer pair for amplifying the 16s rRNA V7-V9 segment; The first primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 2; The second primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 4; The third primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 6; The fourth primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:

8.

2. A reagent for constructing a full-length 16s rRNA high-throughput sequencing library based on the second-generation sequencing technology, characterized in that: Includes primers for library construction; The primer for library construction is formed by sequentially connecting a linker, a custom index and one of the four-plex PCR amplification primers in claim 1.

3. The reagent according to claim 2, characterized in that The linker comprises a linker 1 with a nucleotide sequence as shown in SEQ ID NO:9 and a linker 2 with a nucleotide sequence as shown in SEQ ID NO:10; The connector 1 is used to connect the forward primer in the quadruple PCR amplification primer; The connector 2 is used to connect the reverse primer in the quadruple PCR amplification primer.

4. The reagent according to claim 2, characterized in that The custom index is an oligonucleotide sequence formed by 2 to 4 random nucleotides in length.

5. The reagent according to any one of claims 2 to 4, characterized in that Also included is an amplification reaction master mix and a high-fidelity DNA polymerase.

6. Use of the reagent according to any one of claims 2 to 5 in constructing a high-throughput sequencing library.

7. Use of the reagent according to any one of claims 2 to 5 in high-throughput second-generation sequencing.

8. A method for constructing a full-length 16s rRNA high-throughput sequencing library based on the second-generation sequencing technology, characterized in that: The following steps are involved: Using the DNA of the sample to be sequenced as a template, respectively, and performing quadruple PCR amplification using the quadruple PCR amplification primers described in claim 1 to obtain quadruple PCR amplification products; The quadruple PCR amplification products are mixed, and the obtained PCR product mixture is used as a template to perform Index PCR amplification using index primers adapted to the Illumina platform to obtain Index PCR amplification products; The Index PCR amplification products are mixed to obtain a high-throughput sequencing library.

9. The construction method according to claim 8, characterized in that: The reaction system of the quadruple PCR amplification is 25 μL, specifically including 12.5 μL of 2×PCR mix, 0.5 μL of high-fidelity DNA polymerase, 1 μL of each forward primer and reverse primer, and 4 μL of ddH2O.

10. The construction method according to claim 8 or 9, characterized in that: The reaction procedure of the quadruple PCR amplification is 95°C for 3 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, 25 cycles; 72°C for 5 min.

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