Amplicon library high-throughput sequencing method and system based on dark circulation, and kit
By employing a combined sequencing method with dark cycles, the problems of low efficiency and stability in high-throughput sequencing have been solved, resulting in faster sequencing times, lower costs, and improved signal quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-27
AI Technical Summary
Existing high-throughput sequencing technologies suffer from low sequencing efficiency and low stability in amplicon sequencing, especially in clinical applications, which prolongs the time to obtain valid reports and increases costs and operational complexity.
The high-throughput sequencing method of amplicon libraries using dark cycling skips imaging operations on known fixed segments and only acquires signals in the target variable segments by setting a combination of initial imaging cycle, dark cycle and imaging cycle during the sequencing process.
It significantly shortens sequencing time, reduces instrument wear and reagent costs, improves signal quality, and enhances sequencing stability.
Abstract
Description
Technical Field
[0001] This invention relates to the field of high-throughput sequencing technology, and more particularly to a method and system for high-throughput sequencing of amplicon libraries constructed by dark cycling, and a kit for the above-mentioned tests. Background Technology
[0002] The development of high-throughput sequencing technology has greatly promoted research and applications in fields such as genomics, pathogen diagnostics, and microbial ecology. In particular, amplicon-based targeted sequencing has become an important tool for pathogen detection, microbial community analysis, and genetic variation research. Among these methods, the two-step polymerase chain reaction (PCR) method for constructing amplicon libraries is widely used. In this method, the target region is first amplified using target-specific primers modified with a universal tail sequence at the 5' end. Then, a second PCR step introduces the complete sequencing adapter and sample index. The library constructed using this process has a fixed sequence structure at the start of its read (Read1), typically consisting of the sequencing primer binding region, the universal tail sequence, and the target-specific primer sequence. This sequence is known in advance and highly conserved for all amplicon within the same detection panel; therefore, the amount of information it contains that can be used for subsequent bioinformatics analysis (such as species identification, typing, or mutation site determination) is extremely low.
[0003] Currently, mainstream sequencing-by-synthesis technologies based on reversible terminator methods employ a fixed cyclic workflow during sequencing. Each cycle sequentially performs base incorporation, washing, and optical imaging, failing to differentiate based on the varying information value of sequence regions. This "end-to-end imaging" approach leads to significant efficiency and quality issues. Specifically, a large number of sequencing cycles are consumed in reading these known, low-information, fixed sequences, especially in clinical applications emphasizing rapid turnaround times (such as acute infectious pathogen detection), directly prolonging the time to obtain valid reports. Secondly, consecutive low-diversity sequences at the start of sequencing interfere with the sequencer's initial cluster localization and signal calibration, easily leading to poor focusing and loss of fluorescence signal phase synchronization, thus affecting the sequencing quality of subsequent target variable regions. To compensate for this impact, highly diverse standards (such as PhiX controls) are typically incorporated into the sequencing library. This reduces sequencing throughput, resulting in low sequencing efficiency, while also increasing cost and operational complexity.
[0004] Therefore, there is an urgent need in this field for an innovative amplicon sequencing method, a matching kit, and a system that can significantly shorten sequencing time, reduce operating costs, and improve sequencing stability while ensuring data quality. Summary of the Invention
[0005] To address the technical problems of low sequencing efficiency and low sequencing stability, this invention provides a high-throughput sequencing method for amplicon libraries based on dark cycling. The specific technical solution of this invention is as follows: Firstly, a high-throughput sequencing method for amplicon libraries based on dark cycling is provided, comprising the following steps:
[0006] Step S1: Provide an amplicon library, wherein the amplicon in the amplicon library includes a first fixed segment and a target variable segment in the Read1 sequencing direction, and the first fixed segment includes a universal tail sequence and a target-specific primer binding region;
[0007] Step S2: Sequencing the amplicon library on a high-throughput sequencer based on reversible terminator sequencing chemistry, performing the following operations during sequencing in the Read1 direction:
[0008] a. Set the number N of initial imaging cycles to be executed, wherein the initial imaging cycles perform base incorporation and imaging to complete cluster localization, focusing and / or signal correction, wherein N is a preset positive integer;
[0009] b. Set the number M of dark cycles to be executed immediately following the initial imaging cycle, wherein the dark cycles correspond to a plurality of sequencing cycles of at least a portion of the first fixed segment, wherein the dark cycles perform base incorporation and washing but do not perform imaging operations, wherein M is a preset positive integer;
[0010] c. Set a sequencing cycle corresponding to the target variable segment to execute an imaging cycle, wherein the imaging cycle performs base incorporation, washing and imaging to acquire signals for sequence analysis;
[0011] Step S3: Based on the sequence signal of the target variable segment collected in operation c of step S2, perform species identification, typing and / or site analysis on the sample to be tested.
[0012] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the amplicon library is constructed using a two-step PCR method, including:
[0013] Step S1.1, First step PCR:
[0014] The target nucleic acid was amplified using primers containing the first universal tail sequence UT1 and the second universal tail sequence UT2, respectively.
[0015] Step S1.2, Second step PCR:
[0016] The product of the first step PCR was amplified using primers containing sequencing adapters, external barcodes, and sequences complementary to UT1 and UT2 to obtain the amplicon library.
[0017] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the amplicon structure in the Read1 direction includes, from the 5' end to the 3' end, the following in sequence: a heterogeneous spacer region, a universal tail region, a target-specific primer region, and the target variable segment.
[0018] Further preferably, the heterogeneous spacer region contains 3 to 10 random bases.
[0019] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the value of N is in the range of 4 to 15, the total number of dark cycles is 5 to 30, and the base positions covered by the dark cycles cover at least 80% of the total length of the first fixed segment.
[0020] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the high-throughput sequencer is NextSeq, MiniSeq, or a sequencing platform that is chemically compatible with NextSeq or MiniSeq sequencing.
[0021] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the amplicon library is used for pathogen-targeted sequencing (tNGS), and the target variable region includes species identification regions and / or drug resistance-related sites of bacteria, fungi, viruses and / or parasites.
[0022] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the amplicon library is used for microbial community analysis, and the target variable segment is at least one amplicon and / or ITS segment from the V1 to V9 segments of the bacterial 16S rRNA gene.
[0023] Secondly, a kit for implementing the above-described high-throughput sequencing method is provided, comprising:
[0024] (1) Primer pair for the first step PCR, wherein the primer pair includes target-specific primers respectively carrying the first universal tail sequence UT1 and the second universal tail sequence UT2;
[0025] (2) The second step of PCR uses library construction primer pairs, which include primers with sequencing adapters, external barcodes and sequences complementary to UT1 and UT2;
[0026] The lengths of UT1 and UT2 are independently 18 to 30 nucleotides, such that the amplicon library constructed by the kit forms a fixed sequence region at the front end in the Read1 direction, at least a portion of which is configured to skip the dark cycle of imaging during sequencing.
[0027] Thirdly, an amplicon library sequencing system is provided, comprising:
[0028] (1) A high-throughput sequencer for performing cyclic sequencing of amplicon libraries based on reversible terminal terminators;
[0029] (2) A sequencing control module is configured to control the high-throughput sequencer to perform the following operations in the Read1 direction according to a preset formula: execute an initial imaging cycle of number N, wherein the initial imaging cycle performs base incorporation and imaging; then execute a dark cycle of number M corresponding to the universal tail region and the target specific primer region in the amplicon library, wherein the dark cycle performs base incorporation and washing but does not perform imaging operations; finally execute an imaging cycle corresponding to the target variable segment, wherein the imaging cycle performs base incorporation, washing and imaging operations; wherein N and M are preset positive integers;
[0030] (3) Data analysis module, used for species identification, typing and / or site analysis based on sequencing data collected from the target variable region.
[0031] Compared with the prior art, the present invention has the following technical effects:
[0032] This invention provides a high-throughput sequencing method that first provides an amplicon library containing a first fixed region and a target variable region at the front end of the read length. The first fixed region includes a universal tail sequence and a target-specific primer binding region. Then, the amplicon library is sequenced through the following steps: First, an initial imaging cycle of number N is performed to complete cluster localization and signal correction. Subsequently, in the portion corresponding to the first fixed region, a dark cycle of number M is performed, skipping imaging and only performing base incorporation and washing. Finally, imaging is resumed upon entering the target variable region, and valid sequence signals are acquired for analysis. This invention, by systematically integrating the dark cycle into the sequencing workflow, effectively eliminates invalid imaging of low-information sequences in known target-specific primer binding regions. It significantly shortens sequencing time, reduces instrument wear and reagent costs, and improves signal quality issues caused by low-diversity starting sequences, while ensuring sequencing quality of the target region in the target variable region. Detailed Implementation
[0033] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.
[0034] In one embodiment, a high-throughput sequencing method for amplicon libraries based on dark cycling is provided, which includes the following steps:
[0035] Step S1: Provide an amplicon library, wherein the amplicon in the amplicon library includes a first fixed segment and a target variable segment in the Read1 sequencing direction, and the first fixed segment includes a universal tail sequence and a target-specific primer binding region;
[0036] Step S2: Sequencing the amplicon library on a high-throughput sequencer based on reversible terminator sequencing chemistry, performing the following operations during sequencing in the Read1 direction:
[0037] a. Set the number N of initial imaging cycles to be executed, wherein the initial imaging cycles perform base incorporation and imaging to complete cluster localization, focusing and / or signal correction, wherein N is a preset positive integer;
[0038] b. Set the number M of dark cycles to be executed immediately following the initial imaging cycle, wherein the dark cycles correspond to a plurality of sequencing cycles of at least a portion of the first fixed segment, wherein the dark cycles perform base incorporation and washing but do not perform imaging operations, wherein M is a preset positive integer;
[0039] c. Set a sequencing cycle corresponding to the target variable segment to execute an imaging cycle, wherein the imaging cycle performs base incorporation, washing and imaging to acquire signals for sequence analysis;
[0040] Step S3: Based on the sequence signal of the target variable segment collected in operation c of step S2, perform species identification, typing and / or site analysis on the sample to be tested.
[0041] This invention provides a high-throughput sequencing method that first provides an amplicon library containing a first fixed region and a target variable region at the front end of the read length. The first fixed region includes a universal tail sequence and a target-specific primer binding region. Then, the amplicon library is sequenced through the following steps: First, an initial imaging cycle of number N is performed to complete cluster localization and signal correction. Subsequently, in the portion corresponding to the first fixed region, a dark cycle of number M is performed, skipping imaging and only performing base incorporation and washing. Finally, imaging is resumed upon entering the target variable region, and valid sequence signals are acquired for analysis. This invention, by systematically integrating the dark cycle into the sequencing workflow, effectively eliminates invalid imaging of low-information sequences in known target-specific primer binding regions. It significantly shortens sequencing time, reduces instrument wear and reagent costs, and improves signal quality issues caused by low-diversity starting sequences, while ensuring sequencing quality of the target region in the target variable region.
[0042] As a preferred embodiment, the amplicon library is constructed using a two-step PCR method, including:
[0043] Step S1.1, First step PCR:
[0044] The target nucleic acid was amplified using primers containing the first universal tail sequence UT1 and the second universal tail sequence UT2, respectively.
[0045] Step S1.2, Second step PCR:
[0046] The product of the first step PCR was amplified using primers containing sequencing adapters, external barcodes, and sequences complementary to UT1 and UT2 to obtain the amplicon library.
[0047] As a preferred embodiment of the above-mentioned high-throughput sequencing method, the amplicon structure in the Read1 direction includes, from the 5' end to the 3' end, the following in sequence: a heterogeneous spacer region, a universal tail region, a target-specific primer region, and the target variable segment.
[0048] Further preferably, the heterogeneous spacer region contains 3 to 10 random bases.
[0049] In a preferred embodiment, the value of N is 4 to 15, the total number of dark cycles is 5 to 30, and the base positions covered by the dark cycles cover at least 80% of the total length of the first fixed segment.
[0050] As a preferred embodiment, the high-throughput sequencer is NextSeq, MiniSeq, or a sequencing platform that is chemically compatible with NextSeq or MiniSeq sequencing.
[0051] As a preferred embodiment, the amplicon library is used for pathogen-targeted sequencing (tNGS), and the target variable region includes species identification regions and / or drug resistance-related sites for bacteria, fungi, viruses and / or parasites.
[0052] As a preferred embodiment, the amplicon library is used for microbial community analysis, and the target variable segment is at least one amplicon and / or ITS segment from the V1 to V9 segments of the bacterial 16S rRNA gene.
[0053] In one embodiment, based on the above-described high-throughput sequencing method, a kit for implementing the above-described high-throughput sequencing method is provided, comprising:
[0054] (1) Primer pair for the first step PCR, wherein the primer pair includes target-specific primers respectively carrying the first universal tail sequence UT1 and the second universal tail sequence UT2;
[0055] (2) The second step of PCR uses library construction primer pairs, which include primers with sequencing adapters, external barcodes and sequences complementary to UT1 and UT2;
[0056] The lengths of UT1 and UT2 are independently 18 to 30 nucleotides, such that the amplicon library constructed by the kit forms a fixed sequence region at the front end in the Read1 direction, at least a portion of which is configured to skip the dark cycle of imaging during sequencing.
[0057] In one embodiment, based on the above-described high-throughput sequencing method, an amplicon library sequencing system is provided, comprising:
[0058] (1) A high-throughput sequencer for performing cyclic sequencing of amplicon libraries based on reversible terminal terminators;
[0059] (2) A sequencing control module is configured to control the high-throughput sequencer to perform the following operations in the Read1 direction according to a preset formula: execute an initial imaging cycle of number N, wherein the initial imaging cycle performs base incorporation and imaging; then execute a dark cycle of number M corresponding to the universal tail region and the target specific primer region in the amplicon library, wherein the dark cycle performs base incorporation and washing but does not perform imaging operations; finally execute an imaging cycle corresponding to the target variable segment, wherein the imaging cycle performs base incorporation, washing and imaging operations; wherein N and M are preset positive integers;
[0060] (3) Data analysis module, used for species identification, typing and / or site analysis based on sequencing data collected from the target variable region.
[0061] As can be seen from the above description, the high-throughput sequencing method of the present invention is applicable to microbial amplicon sequencing applications such as pathogen tNGS and 16S / ITS. To make the present invention clearer and easier to understand, the following examples, in conjunction with different sequencing targets, further describe the present invention.
[0062] Example 1
[0063] This embodiment uses respiratory pathogen targeted sequencing as an example to provide a pathogen tNGS amplicon library sequencing method based on dark cycling, illustrating the specific implementation process of the method of the present invention. The specific steps are as follows:
[0064] I. Sample and Nucleic Acid Extraction
[0065] 200 μL of clinical bronchoalveolar lavage fluid (BALF) was selected. Using a commercial nucleic acid extraction kit, following the instructions, 30–50 μL of total nucleic acid was obtained and used as the sample to be tested, stored at -20℃ for later use. In this example, the Qiagen QIAamp® DNA Mini Kit (250) was used for nucleic acid extraction.
[0066] II. Step 1 PCR
[0067] This step amplifies the nucleic acid in the sample by using primer sequences that include a universal tail and target-specific primers for the sequence to be tested.
[0068] By leveraging the highly similar sequences (conserved regions) among different pathogen species, universal target-specific primers are designed to ensure the capture of a broad spectrum of pathogen targets. Simultaneously, this embodiment introduces a universal tail while designing universal target-specific primers. Specifically, multiple specific primers are designed for common respiratory pathogens, including influenza virus, RSV, adenovirus, and Streptococcus pneumoniae, with each primer being 18–24 nt in length. A universal tail sequence UT1 or UT2 is attached to the 5' end of each specific primer. Both UT1 and UT2 are 22 nt in length. Specifically, the UT1 sequence is: 5'-ACACTGACGACATGGTTCTACA-3'; and the UT2 sequence is: 5'-TACGGTAGCAGAGACTTGGTCT-3'.
[0069] More specifically, optionally, three random bases NNN are added between UT1, UT2 and the specific primer as a heterogeneous spacer, with the structure: 5'-UT1–NNN–specific primer F, 5'-UT2–NNN–specific primer R.
[0070] Based on the above, the reaction system for the first step of PCR (25 μL) included: 12.5 μL of 2× PCR Master Mix; 1.0 μL of forward multiple primer mixture containing UT1 (10 μM each); 1.0 μL of reverse multiple primer mixture containing UT2 (10 μM each); 2–5 μL of template nucleic acid; and nuclease-free water to a final volume of 25 μL. PCR reaction conditions were: 98 ℃ pre-denaturation for 2 min; 25–30 cycles of 98 ℃ for 10 s, 60 ℃ for 30 s, and 72 ℃ for 30 s; extension at 72 ℃ for 5 min; and incubation at 4 ℃. After the reaction, the sample was purified using 0.8×–1.0× magnetic beads, with an elution volume of 20 μL, which was used as the template for the second step of PCR.
[0071] III. Second Step PCR
[0072] This step amplifies the product from the first step of PCR using primer sequences containing adapters and external barcodes.
[0073] Library construction primer pairs were designed as follows: Forward library construction primers: 5'-P5 adapter + i5 barcode (8 nt) + Read1 sequencing primer binding region + UT1 complementary sequence - 3'; Reverse library construction primers: 5'-P7 adapter + i7 barcode (8 nt) + Read2 sequencing primer binding region + UT2 complementary sequence - 3'. Second reaction system (50 μL): 2× PCR Master Mix 25 μL; Forward library construction primers (10 μM) 1.0 μL; Reverse library construction primers (10 μM) 1.0 μL; First PCR product 5–10 μL; Nuclease-free water to a final volume of 50 μL. PCR conditions: 98 ℃ pre-denaturation for 2 min; 98 ℃ for 10 s, 60 ℃ for 30 s, 72 ℃ for 30 s, 10–14 cycles; 72 ℃ extension for 5 min, 4 ℃ incubation. The product was purified with 0.8×–1.0× magnetic beads, and 20 μL was eluted to obtain an amplicon library suitable for PCR.
[0074] IV. Quantitative Library Application and Computer Testing
[0075] The library was quantified using qPCR or quantitative real-time sequencing and diluted to 1.0–2.0 nM according to the sequencing platform requirements. After mixing, the library was loaded into the NextSeq high-throughput flow cell according to the manufacturer's instructions, using the single-end 75 bp (SE75) mode. The amplicon structure in the Read1 direction was: 5'-P5 adapter / Read1 primer binding site-NNN (3 bp spacer)-UT1 (22 bp)-specific primer F (approximately 20 bp)-target variable region (≥100 bp)-3'.
[0076] V. Sequencing Recipe and Dark Cycle Setup
[0077] Sequencing mode: SE75. Based on the above structure, the first 3 bp spacer of Read1 and part of the universal tail are used for focusing and cluster localization. In this embodiment, the following settings are configured: Cycles 1-8: Normal imaging cycles (including NNN spacer + the first 5 bp of UT1); Cycles 9-20: Dark cycles, only base incorporation and washing are performed, no imaging is performed, corresponding to the remaining part of UT1 and specific primer F; Cycles 21-75: Recovery imaging, corresponding to the target variable region. In the sequencing control software, the above imaging / dark cycle combination is set by a custom recipe, keeping the total number of SBS cycles constant at 75. Other sequencing parameters (such as loading concentration, cluster density, PhiX incorporation ratio, etc.) are set according to the platform's recommended range. Through the above settings in this embodiment, the PhiX incorporation ratio is controlled at 0-1%, which effectively reduces the PhiX incorporation ratio.
[0078] VI. Data Analysis
[0079] For the acquired FASTQ data, only the 21st to 75th bases of Read1 were retained as valid sequences. Using proprietary or publicly available alignment and species interpretation procedures, the reads were aligned to the pathogen reference database, and the number of reads, coverage, and drug resistance sites for each target were calculated. Compared with the data obtained using the conventional definition (no dark cycle, 75 consecutive imaging cycles of Read1), in this embodiment, the Q30 value of the insert region, read distribution, positive detection rate, and drug resistance site consistency did not decrease significantly, while the sequencing run time was shortened by about 50 minutes due to the reduction of 12 imaging cycles.
[0080] Example 2
[0081] This embodiment uses the application of bacterial 16S rRNA in microbial community analysis as an example to provide a 16S rRNA amplicon sequencing method based on dark cycling. Specifically, it includes the following steps:
[0082] I. Construction of 16S amplicon library
[0083] 200 mg of feces was selected, and total DNA was extracted using conventional methods.
[0084] Step 1 PCR: A 16S V3-V4 region primer pair with universal tails was used: Fwd: 5'-UT1-NNN–16S_V3F-3'; Rev: 5'-UT2-NNN-16S_V4R-3'; where UT1 and UT2 are 22 nt universal tails, and 16S_V3F / V4R are 16S amplification primers. The reaction system and PCR conditions were the same as in Step 1 PCR of Example 1. The PCR product was purified with magnetic beads and used as the template for Step 2 PCR.
[0085] Step 2 PCR: Amplification was performed using primers containing P5 / P7 adapters, external barcodes, and UT1' / UT2' complementary sequences, under the same conditions as Step 2 PCR in Example 1. After purification, a 16S amplicon library was obtained, with a library structure similar to that in Example 1.
[0086] The partial sequences of bacterial 16S rRNA genes are very similar, which allows for the identification and amplification of almost all bacteria using a universal set of "PCR primers"—target-specific primers for the sequence to be tested, as described above. A portion of their 16S rRNA gene sequence is a variable region, which varies significantly among different bacterial species, allowing for the differentiation of different bacterial species.
[0087] II. Sequencing and Dark Cycling Settings
[0088] Sequencing was performed using the MiSeq or MiniSeq platform in paired-end sequencing mode at 2×150 bp. Dark cycling was configured in the Read1 direction: Cycles 1–10: normal imaging (including the NNN spacer and part of UT1); Cycles 11–25: dark cycling (corresponding to the remaining UT1 and the 16S_V3F primer region); Cycles 26 and beyond: normal imaging, corresponding to the 16S V3-V4 variant region. Read2 could use the conventional imaging mode, or, as needed, a similar dark cycling configuration could be applied to a fixed segment at its front.
[0089] III. 16S Analysis Process
[0090] Quality control was performed on Read1 and Read2, and they were assembled using conventional methods to remove connector sequences. Noise removal, feature sequence clustering, and species annotation were performed using QIIME2, DADA2, or a self-built analysis workflow. Alpha diversity, beta diversity, and abundance of each species among the samples were calculated.
[0091] Compared with 16S sequencing without dark cycling, this embodiment shortens the single run time by about 1 hour by skipping about 15 imaging cycles of the universal tail + primer region. At the same time, there are no significant differences in indicators such as OTU / ASV number, species composition and abundance distribution, proving that the method of the present invention is also applicable to 16S amplicon sequencing.
[0092] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.
[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. A high-throughput sequencing method for amplicon libraries based on dark cycling, characterized in that: Includes the following steps: Step S1: Provide an amplicon library, wherein the amplicon in the amplicon library includes a first fixed segment and a target variable segment in the Read1 sequencing direction, and the first fixed segment includes a universal tail sequence and a target-specific primer binding region; Step S2: Sequencing the amplicon library on a high-throughput sequencer based on reversible terminator sequencing chemistry, performing the following operations during sequencing in the Read1 direction: a. Set the number N of initial imaging cycles to be executed, wherein the initial imaging cycles perform base incorporation and imaging to complete cluster localization, focusing and / or signal correction, wherein N is a preset positive integer; b. Set the number M of dark cycles to be executed immediately following the initial imaging cycle, wherein the dark cycles correspond to a plurality of sequencing cycles of at least a portion of the first fixed segment, wherein the dark cycles perform base incorporation and washing but do not perform imaging operations, wherein M is a preset positive integer; c. Set a sequencing cycle corresponding to the target variable segment to execute an imaging cycle, wherein the imaging cycle performs base incorporation, washing and imaging to acquire signals for sequence analysis; Step S3: Based on the sequence signal of the target variable segment collected in operation c of step S2, perform species identification, typing and / or site analysis on the sample to be tested.
2. The high-throughput sequencing method as described in claim 1, characterized in that: The amplicon library was constructed using a two-step PCR method, including: Step S1.1, First step PCR: The target nucleic acid was amplified using primers containing the first universal tail sequence UT1 and the second universal tail sequence UT2, respectively. Step S1.2, Second step PCR: The product of the first step PCR was amplified using primers containing sequencing adapters, external barcodes, and sequences complementary to UT1 and UT2 to obtain the amplicon library.
3. The high-throughput sequencing method as described in claim 1 or 2, characterized in that: The amplicon structure in the Read1 direction includes, from the 5' end to the 3' end, the following components in sequence: a heterogeneous spacer region, a universal tail region, a target-specific primer region, and the target variable segment.
4. The high-throughput sequencing method as described in claim 3, characterized in that: The heterogeneous spacer region contains 3 to 10 random bases.
5. The high-throughput sequencing method as described in claim 1, characterized in that: The value of N ranges from 4 to 15, the total number of dark cycles is 5 to 30, and the base positions covered by the dark cycles cover at least 80% of the total length of the first fixed segment.
6. The high-throughput sequencing method as described in claim 1, characterized in that: The high-throughput sequencer is NextSeq, MiniSeq, or a sequencing platform that is chemically compatible with NextSeq or MiniSeq sequencing.
7. The high-throughput sequencing method as described in claim 1, characterized in that: The amplicon library is used for pathogen-targeted sequencing (tNGS), and the target variable region contains species identification regions and / or drug resistance-related sites for bacteria, fungi, viruses and / or parasites.
8. The high-throughput sequencing method as described in claim 1, characterized in that: The amplicon library is used for microbial community analysis, and the target variable segment is at least one amplicon and / or ITS segment from the V1 to V9 segments of the bacterial 16S rRNA gene.
9. A kit for implementing the method according to any one of claims 1 to 8, characterized in that: include: (1) Primer pair for the first step PCR, wherein the primer pair includes target-specific primers respectively carrying the first universal tail sequence UT1 and the second universal tail sequence UT2; (2) The second step of PCR uses library construction primer pairs, which include primers with sequencing adapters, external barcodes and sequences complementary to UT1 and UT2; The lengths of UT1 and UT2 are independently 18 to 30 nucleotides, such that the amplicon library constructed by the kit forms a fixed sequence region at the front end in the Read1 direction, at least a portion of which is configured to skip the dark cycle of imaging during sequencing.
10. An amplicon library sequencing system, characterized in that: include: (1) A high-throughput sequencer for performing cyclic sequencing of amplicon libraries based on reversible terminal terminators; (2) A sequencing control module is configured to control the high-throughput sequencer to perform the following operations in the Read1 direction according to a preset formula: execute an initial imaging cycle of number N, wherein the initial imaging cycle performs base incorporation and imaging; then execute a dark cycle of number M corresponding to the universal tail region and the target specific primer region in the amplicon library, wherein the dark cycle performs base incorporation and washing but does not perform imaging operations; finally execute an imaging cycle corresponding to the target variable segment, wherein the imaging cycle performs base incorporation, washing and imaging operations; wherein N and M are preset positive integers; (3) Data analysis module, used for species identification, typing and / or site analysis based on sequencing data collected from the target variable region.