Primer combination for detecting POLE gene multi-hotspot mutation, kit and library construction method

Abstract

The invention discloses a primer combination, a kit and a library construction method for detecting POLE gene multi-hotspot mutation, the primer combination is used for targeted amplification of POLE gene mutation sites, and the primer combination comprises P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F and A456P. The kit can detect 11 mutation sites of the POLE gene at the same time, directly reflects mutation conditions of the sites, and is used for endometrial cancer POLE molecular typing.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular to a primer combination, a kit and a library construction method for detecting multiple hotspot mutations in POLE. Background Art

[0002] The POLE gene belongs to the DNA polymerase B family and is a catalytic subunit of DNA polymerase ε. It possesses both 5'→3' DNA polymerase and 3'→5' exonuclease activities and plays a crucial role in DNA replication and proofreading. The exonuclease domain of this gene encompasses exons 9–14. Mutations in this gene can lead to uncorrected new mutations generated during DNA synthesis, potentially contributing to tumorigenesis. Whole-exome and whole-genome sequencing studies have confirmed that approximately 600 human tumors are associated with POLE gene mutations, with endometrial cancer having the highest mutation rate, reaching 10%. Detection of the 11 pathogenic variants of the POLE gene in endometrial cancer (EC) is crucial to identify women with a favorable prognosis and reduce overtreatment.

[0003] In 2020, over 417,000 women worldwide were diagnosed with endometrial cancer (EC), making it the sixth most common cancer in women. Its incidence has increased dramatically over the past few decades, and while most patients require surgery alone, many still require adjuvant therapy. Both domestic and international guidelines recommend POLE mutation testing for patients diagnosed with endometrial cancer. Guidelines such as the "Guidelines for the Diagnosis and Treatment of Endometrial Cancer (2021 Edition)" and the "NCCN Clinical Practice Guidelines for Uterine Oncology (2022V1)" recommend POLE gene testing for patients with endometrial adenocarcinoma. They recommend combining MMR / MSI status and p53 status to categorize endometrial cancer into four molecular subtypes: POLE hypermutation (POLEmut), mismatch repair gene deficiency (MMRd), nonspecific molecular profile (NSMP), and p53 mutation (p53abn). Although POLEmut (POLEmut) accounts for only 8-10% of all endometrial cancers, studies have identified 11 pathogenic hotspot mutations in the exonuclease domain of DNA polymerase. Endometrial cancer patients with these mutations have a favorable clinical prognosis and a very low risk of recurrence. Over 95% of POLEmut ECs carry mutations in P286R, S297F, V411L-T / C, A456P, or S459F, while approximately 4.4% exhibit mutations in other domains (M295R, F367S, D368Y, L424I, P436R, and M444K).

[0004] Given the above situation, there is a significant demand for POLE gene mutation detection, but current detection methods have limitations. Common methods for detecting gene mutations include fluorescent quantitative PCR, gene chip methods, Sanger sequencing, and high-throughput sequencing. Fluorescent quantitative PCR and gene chip methods can only detect one or several known mutation sites, while Sanger sequencing can only sequence a specific region of a sample at a time. Common disadvantages of these methods include being unable to detect unknown mutation sites, low detection efficiency, difficult results to interpret, poor reproducibility, and high false positive and false negative results. A highly sensitive, economical, and convenient method for detecting POLE gene mutations is urgently needed.

[0005] To overcome this, we developed and validated a rapid, low-cost primer combination and kit for the simultaneous detection of 11 POLE pathogenic hotspots in a single assay by targeted NGS sequencing for clinical endometrial cancer. Summary of the Invention

[0006] According to the first aspect, in one embodiment, a primer combination is provided, which is used for targeted amplification of the POLE gene mutation site, including: P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F, and A456P.

[0007] According to the second aspect, one embodiment provides a kit comprising the primer combination of the first aspect.

[0008] According to the third aspect, an embodiment provides a library construction method, comprising:

[0009] an adapter ligation step, comprising ligating an adapter with a molecular tag to a nucleic acid sample to obtain a sample ligated with an adapter;

[0010] The first round of nested PCR amplification step includes amplifying the sample connected to the adapter using outer primers to obtain a first round of amplification products;

[0011] The second round amplification step of the nested PCR includes using inner primers to amplify the sample connected to the adapter to obtain a second round amplification product.

[0012] According to the primer combination, kit and library construction method for targeted amplification of POLE gene mutation sites in the above embodiment, the present invention can simultaneously detect 11 mutation sites of POLE and directly reflect the mutation status of the relevant mutation sites.

[0013] In one embodiment, the molecular tagging and nested multiplex PCR employed in the present invention effectively eliminates false positives introduced during the PCR process, improving the detection limit for samples with low-frequency mutations, and consistently detecting low-frequency variants as low as 0.1%. This method enables efficient and sensitive detection of POLE gene mutations while reducing costs and simplifying the protocol. It can be used for the detection of POLE mutations and for the molecular typing of endometrial cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A flowchart for library construction in one embodiment. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core of the present application being overwhelmed by excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. They can fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

[0016] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0017] The serial numbers assigned to the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0018] High-throughput sequencing (NGS) is an internationally advanced new sequencing technology capable of sequencing hundreds of thousands to millions of DNA molecules simultaneously, significantly increasing sequencing throughput. Furthermore, with the rapid development of bioinformatics, integrated multi-site detection and analysis can simultaneously analyze and analyze gene loci closely associated with tumors, identifying genetic mutations in patients.

[0019] There are two main types of mainstream high-throughput sequencing technologies: large and comprehensive whole-genome / whole transcriptome library sequencing, and small and precise targeted library sequencing. While whole-genome and whole-exome sequencing methods can meet clinical needs in terms of throughput, their high cost prevents widespread clinical application. Therefore, targeted library sequencing, with its advantages of low cost, high data utilization, and flexible combination, is playing a positive role in clinical disease diagnosis and treatment, as well as in tumor genetic testing.

[0020] Library construction methods within targeted sequencing technology are categorized into hybridization capture and multiplex PCR. Targeted multiplex PCR reduces the library construction process by simply adding multiple primers to simultaneously amplify the target sequence, significantly shortening the construction time. Multiplex PCR is a PCR technique that uses multiple primer pairs in a single reaction system to simultaneously amplify different gene fragments from the same DNA sample. By analyzing the sequences of these different gene fragments, it is possible to determine whether deletions, insertions, or point mutations exist within the gene.

[0021] According to the first aspect, in one embodiment, a primer combination is provided, which is used for targeted amplification of the POLE gene hotspot mutation site, including: P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F, A456P.

[0022] In one embodiment, the primer combination comprises an outer primer and an inner primer.

[0023] In one embodiment, the outer primers comprise at least one of the sequences with sequence number 2n among the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥0.

[0024] In one embodiment, the inner primer comprises at least one of the nucleotide sequences shown in SEQ ID No. 1 to 20, with sequence number 2n+1, where n is an integer ≥0.

[0025] In one embodiment, the primer combination is used to detect POLE mutations.

[0026] According to the second aspect, in one embodiment, a kit is provided, comprising the primer combination described in the first aspect.

[0027] In one embodiment, it further comprises at least one of a linker with a molecular tag, a library pretreatment reagent, a targeted amplification reagent, a library amplification reagent, an I5 tag, an I7 tag, and a purification reagent.

[0028] In one embodiment, the library pre-treatment reagent comprises at least one of a terminal "A" adding reagent and an adapter ligation reagent.

[0029] In one embodiment, the purification reagent comprises magnetic beads, including but not limited to KRS™ Pure Beads or Agencourt AMPure XP magnetic beads.

[0030] According to the third aspect, in one embodiment, a library construction method is provided, comprising:

[0031] an adapter ligation step, comprising ligating an adapter with a molecular tag to a nucleic acid sample to obtain a sample ligated with an adapter;

[0032] The first round of nested PCR amplification step includes amplifying the sample connected to the adapter using outer primers to obtain a first round of amplification products;

[0033] The second round amplification step of the nested PCR includes using inner primers to amplify the sample connected to the adapter to obtain a second round amplification product.

[0034] In one embodiment, the outer primers comprise at least one of the sequences with sequence number 2n among the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥0.

[0035] In one embodiment, the inner primer comprises at least one of the nucleotide sequences shown in SEQ ID No. 1 to 20, with sequence number 2n+1, where n is an integer ≥0.

[0036] In one embodiment, in the first round amplification step of the nested PCR, the reaction system contains primers that are specifically reverse complementary to at least a portion of the sequence in the molecularly tagged adapter.

[0037] In one embodiment, in the first round amplification step of the nested PCR, the reaction system further contains a targeted amplification reagent.

[0038] In one embodiment, in the second round of nested PCR amplification step, the reaction system contains primers that are specifically reverse-complementary paired with at least a portion of the sequence in the molecularly tagged adapter.

[0039] In one embodiment, in the first round amplification step of the nested PCR, the reaction system further contains a targeted amplification reagent.

[0040] In one embodiment, a purification step is further included, including performing magnetic bead purification on the second-round amplification product to obtain a purified product.

[0041] In one embodiment, the method further comprises a library amplification step, comprising amplifying the purified product to obtain a library amplification product.

[0042] In one embodiment, in the library amplification step, the reaction system contains at least one of a library amplification reagent, an I5 tag, and an I7 tag.

[0043] In one embodiment, the method further includes a purification step, which includes performing magnetic bead purification on the amplified product of the library to obtain a library that can be used for sequencing.

[0044] In one embodiment, in the adapter ligation step, the nucleic acid sample is a sample that has undergone end repair and "A" addition reaction.

[0045] According to the fourth aspect, in one embodiment, a library prepared by the library construction method described in the third aspect is provided.

[0046] In one embodiment, the present invention incorporates unique molecular identifiers (UMIs) during multiplex PCR to help correct errors during amplification and sequencing. For UMI-labeled reads, sequencing reads with the same UMI can be grouped together during data analysis. Correction of the grouped data yields a final single-stranded consensus sequence (SSCS, which removes duplicate sequences based on the UMI sequence and its alignment to the genome, forming a single-stranded consensus sequence). This allows for accurate mutation results and reduces background noise.

[0047] In one embodiment, the present invention provides a multiplex PCR-specific primer, kit, and detection method for detecting POLE gene mutations based on targeted high-throughput sequencing technology, which can simultaneously detect mutations in multiple genes and multiple sites, and effectively improve detection efficiency and accuracy, while reducing costs and simplifying operating steps.

[0048] In one embodiment, the present invention provides a PCR primer pair, kit, and method for detecting POLE gene mutations by targeted high-throughput sequencing based on molecular tagging and nested multiplex PCR. The molecular tagging and nested multiplex PCR technology employed can effectively eliminate false positives introduced during the PCR process, improving detection sensitivity and accuracy.

[0049] In one embodiment, the present invention provides a primer pair for high-throughput targeted sequencing of POLE gene mutations based on molecular tagging and nested multiplex PCR. The nucleotide sequences of the two-round nested multiplex PCR primers are shown in Table 1, SEQ ID No. 1 to SEQ ID No. 20. The outer primers are used in the first round of nested PCR amplification, and the inner primers are used in the second round of nested PCR amplification. The mutation sites are P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F, and A456P. The mutation can be a substitution, insertion, and / or deletion of one or more bases.

[0050] The downstream primer in the outer primer is the sequence numbered 2n among the nucleotide sequences shown in SEQ ID Nos. 11 to 20, where n is an integer ≥ 1. For example, the nucleotide sequences shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, and SEQ ID No. 10 are the upstream primers of the outer primer. The nucleotide sequences shown in SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, and SEQ ID No. 20 are the downstream primers of the outer primer.

[0051] The inner primer is specifically a sequence numbered 2n+1 among the nucleotide sequences set forth in SEQ ID Nos. 1 to 20, where n is an integer ≥ 0. For example, the nucleotide sequences set forth in SEQ ID Nos. 1, 3, 5, 7, and 9 are upstream primers of the inner primer. The nucleotide sequences set forth in SEQ ID Nos. 11, 13, 15, 17, and 19 are downstream primers of the inner primer.

[0052] In one embodiment, the specific primers set forth in SEQ ID No. 1 to SEQ ID No. 20 of the present invention are sequences specifically designed to target five POLE gene mutation sites. Specifically, they can amplify regions encompassing 11 POLE gene mutation sites, including P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F, and A456P. Table 2 lists the starting locations of the target regions on the chromosomes for the amplified fragments. High-throughput sequencing is performed to obtain sequence information for the amplified products, thereby enabling the detection of mutations at these sites in the sample. Since the target regions are all detected, a two-round nested multiplex amplification approach is employed based on the principles of multiplex PCR amplification and library construction to ensure specificity and enhance stability. The primers shown in SEQ ID No. 1 to SEQ ID No. 20 used in the present invention can use a two-round nested multiplex PCR amplification technology. The first round of multiplex PCR reaction effectively amplifies the target sequence in one tube at the same time. After purification of the PCR products, the second round of multiplex PCR reaction further effectively amplifies the target sequence in one tube at the same time. After purification of the PCR products, post-PCR reaction and further purification, the resulting PCR products are used to prepare sequencing libraries and are subjected to high-throughput sequencing on a machine. The constructed sequencing libraries are qualified after being detected by Qubit 3.0 and are suitable for various high-throughput sequencing platforms such as BGI and Illumina.

[0053] Table 1 Nucleotide sequences of primers for two rounds of nested multiplex PCR

[0054]

[0055] Table 2 The starting position of the amplified target fragment on the chromosome

[0056]

[0057] In one embodiment, the primer pair sequences of the 11 mutation site regions of the POLE gene based on molecular tagging and nested multiplex PCR for high-throughput targeted sequencing and the regions of the amplified target fragments on the chromosome are also within the scope of protection of the present invention.

[0058] In one embodiment, the present invention provides a kit for detecting POLE mutations by high-throughput targeted sequencing based on molecular tagging and nested multiplex PCR, comprising multiplex PCR primers for two rounds of nesting (outer primers are used in the first round of nested PCR amplification, and inner primers are used in the second round of nested PCR amplification), library pretreatment reagents, targeted amplification reagents, library amplification reagents, I5 tags, I7 tags, and purification magnetic beads.

[0059] In one embodiment, the library pre-treatment reagents include at least one of a terminal "A" reagent and an adapter ligation reagent.

[0060] In one embodiment, the terminal addition "A" reagent includes terminal addition "A" buffer and terminal addition "A" enzyme.

[0061] In one embodiment, the adapter ligation reagent includes an adapter with a UMI, DNA ligase, and a DNA ligation buffer.

[0062] In one embodiment, the targeted amplification reagent includes a high-fidelity DNA polymerase, a PCR buffer, and a dNTP mixture.

[0063] In one embodiment, the kit further includes a primer anchor primer (for Illumina) that is complementary to the specificity of the adapter with the UMI, as well as outer primers used in the first round of nested PCR amplification and inner primers used in the second round of nested PCR amplification.

[0064] In one embodiment, the library amplification reagents include high-fidelity DNA polymerase, PCR buffer, dNTP mixture, and UDI Primer.

[0065] In one embodiment, the purification magnetic beads are used to purify DNA of a target size range from target amplification products and library amplification products.

[0066] Example 1

[0067] The database construction process is as follows Figure 1 The specific steps are as follows:

[0068] 1. A library was constructed and tested using AI-Edigene® POLE p.V411L / POLE p.P286R / POLE p.A456P, the commercially available gDNA standards with known mutations. The mutation frequencies of the commercial gDNA standards were adjusted to four different levels: 10%, 5%, 2.5%, and 1%. After verification by ddPCR, these samples were used for testing. Three replicates were set for each sample. 60 ng of genomic DNA (at a concentration of 2.5 ng / μL) was used for shearing and end-repair using a commercially available kit, and an "A" was added to the 3' end.

[0069] Prepare the reaction system:

[0070] Table 2

[0071]

[0072] Set the PCR instrument parameters as follows:

[0073] Table 3

[0074]

[0075] 2. Dilute the KRS™ UMI Adapter (15 μM, for Illumina) 10-fold to 1 μM / μL using Nuclease-Free Water. Ligate the UMI-containing adapter to the sample with the 3' end "A" added in the previous step.

[0076] Prepare the reaction system:

[0077] Table 4

[0078]

[0079] Set the PCR instrument parameters as follows:

[0080] Table 5

[0081]

[0082] 3. Use KRS™ Pure Beads to purify the adapter-ligated DNA sample from step 2 to obtain 13 μL of eluate.

[0083] 4. Perform the first round of nested PCR amplification using the outer primer pair. Add the outer primer mix pair, targeted amplification reagent, and a KRS™ anchor primer (for Illumina) specifically complementary to the UMI-bearing adapter to the reaction system. Perform the first round of nested PCR amplification of the target region.

[0084] The outer primer pair is specifically a sequence with a sequence number of 2n in the nucleotide sequence shown in SEQ ID No. 1 to 20, where n is an integer ≥0.

[0085] Prepare the following reaction system:

[0086] Table 6

[0087]

[0088] Set the PCR instrument parameters as follows:

[0089] Table 7

[0090]

[0091] 5. Purify the amplicon solution from step 4 using KRS™ Pure Beads or Agencourt AMPure XP magnetic beads to obtain 14 μL of eluate.

[0092] 6. Perform a second round of nested PCR amplification using the inner primer pair. Add the outer primer mix pair, targeted amplification reagent, and a KRS™ anchor primer (for Illumina) specifically complementary to the UMI-bearing adapter to the reaction system. Perform a second round of nested PCR amplification of the target region.

[0093] The inner primer pair is specifically a sequence with a sequence number of 2n+1 in the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥0.

[0094] Prepare the following reaction system:

[0095] Table 8

[0096]

[0097] Set the PCR instrument parameters as follows:

[0098] Table 9

[0099]

[0100] 7. Purify the amplicon solution from step 6 using KRS™ Pure Beads or Agencourt AMPure XP magnetic beads to obtain 15 μL of eluate.

[0101] 8. Add library amplification buffer, I5 tag, I7 tag and double-distilled water to the amplicon liquid obtained in step 7, and perform PCR reaction to add sequencing adapter sequences on both sides of the amplicon.

[0102] Prepare the following reaction system:

[0103] Table 10

[0104]

[0105] Set the PCR instrument parameters as follows:

[0106] Table 11

[0107]

[0108] 9. Purify the amplicon from step 8 using KRS™ Pure Beads or Agencourt AMPure XP magnetic beads to obtain 27 μL of eluate. Use a pipette to aspirate 25 μL of the supernatant and transfer it to a new PCR tube. Label the tube for sequencing. Sequence 1 μL of the library using the Qubit dsDNA HS Assay Kit and record the library concentration. Determine the fragment length using a fragment analyzer using 1 μL of the library.

[0109] 10. After passing the QSEP fragmentation analyzer assay, the DNA was sequenced on the BGI MGISEQ-T7. Three known mutation sites, POLE p.V411L, POLE p.P286R, and POLE p.A456P, were detected reliably across four frequency gradients: 10%, 5%, 2.5%, and 1%.

[0110] Example 2

[0111] Endometrial exfoliated cells were collected from Xiangya Third Hospital. Four samples of gDNA from the POLE-positive samples confirmed in the example of patent authorization number CN116083425B were used. The gDNA concentration was detected by Quibt. Sample processing and on-machine testing were performed according to the operating method of Example 1.

[0112] The test data of the samples are shown in Table 3. All 4 samples that were positive for POLE mutations could be detected, including 3 cases of P286R and 1 case of V411L.

[0113] Table 3 Results of gDNA detection of POLE mutation-positive endometrial exfoliated cells

[0114]

[0115] Table 3

[0116]

[0117] Table 3

[0118]

[0119] Example 3

[0120] Paraffin-embedded section samples of the patients corresponding to Example 2 were obtained from the Department of Pathology of Xiangya Third Hospital. Genomic DNA was extracted using the Tiangen Blood / Cell / Tissue Genomic Kit (Cat. No. DP304). The gDNA concentration was detected by Quibt. Sample processing and on-machine testing were performed according to the operating procedures of Example 1.

[0121] The sample detection data are shown in Table 4. All four tissue samples positive for POLE mutations were detectable, with P286R in three and V411L in one. Mutation sites were consistent between tissue and endometrial exfoliated cells, but the frequencies differed. This analysis may be due to tumor tissue heterogeneity and the proportion of tumor cells in exfoliated cells.

[0122] Table 3 Results of gDNA detection of POLE mutation-positive endometrial exfoliated cells

[0123]

[0124] Table 3

[0125]

[0126] Table 3

[0127]

[0128] The present invention provides an efficient and reliable high-throughput sequencing sequence enrichment method. The 11 pathogenic hotspot mutation sites of the POLE gene have the advantages of high detection throughput and strong specificity, and the detection results have good repeatability and accuracy.

[0129] In one embodiment, the present invention provides a primer pair, kit, and method for detecting POLE gene mutations using high-throughput targeted sequencing based on molecular tagging and nested multiplex PCR. The present invention specifically discloses multiplex PCR primers as shown in SEQ ID No. 1 to SEQ ID No. 20, a kit containing such primers, and a method for using such primers or kit, combined with two rounds of nested PCR amplification, to target and enrich POLE gene fragments for detecting gene mutation sites. The method specifically includes sample DNA extraction, end-addition of A, ligation of single-end adapter sequences containing UMI molecular tags, two rounds of nested PCR amplification to enrich target gene fragments, library quality control, and high-throughput sequencing.

[0130] In one embodiment, the method of the present invention detects gDNA from exfoliated cells of the uterine cavity for endometrial cancer. In another embodiment, the method of the present invention detects gDNA from exfoliated cells of the uterine cavity for patients with endometrial cancer. The present invention can be used for the detection of both FFPE tissue samples and exfoliated cells of the uterine cavity. The detection of exfoliated cells of the uterine cavity and FFPE tissue sites shows good consistency. The use of POLE for preoperative detection of exfoliated cells of the uterine cavity can help doctors preliminarily predict the molecular typing of endometrial cancer in patients, helping to determine more optimal surgical and treatment strategies.

[0131] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A primer combination, characterized in that: The primer combination is used to target and amplify the sites of POLE gene mutations, including: P286R, S297F, M295R, F367S, D368Y, V411L, L424I, P436R, M444K, S459F, and A456P.

2. The primer combination according to claim 1, wherein The primer combination comprises an outer primer and an inner primer; Preferably, the outer primers comprise at least one of the sequences numbered 2n among the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥ 0; Preferably, the inner primer comprises at least one of the sequences with sequence number 2n+1 among the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥ 0; Preferably, the primer combination is used to detect POLE gene mutation.

3. A kit, characterized in that Comprising the primer combination according to claim 1 or 2.

4. The kit according to claim 3, wherein It also includes at least one of a linker with a molecular tag, a library pretreatment reagent, a targeted amplification reagent, a library amplification reagent, an I5 tag, an I7 tag, and a purification reagent.

5. The kit according to claim 4, wherein The library pre-treatment reagents include at least one of a terminal "A" reagent and a linker ligation reagent; Preferably, the purification reagent comprises magnetic beads.

6. A library construction method, characterized in that, include: an adapter ligation step, comprising ligating an adapter with a molecular tag to a nucleic acid sample to obtain a sample ligated with an adapter; The first round of nested PCR amplification step includes amplifying the adapter-connected sample using outer primers to obtain a first round of amplification products; The second round amplification step of the nested PCR includes using inner primers to amplify the sample connected to the adapter to obtain a second round amplification product.

7. The library construction method according to claim 6, wherein The outer primers comprise at least one of the sequences numbered 2n in the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥ 0; Preferably, the inner primer comprises at least one of the sequences with sequence number 2n+1 among the nucleotide sequences shown in SEQ ID No. 1 to 20, where n is an integer ≥0.

8. The library construction method according to claim 6, wherein In the first round of amplification of the nested PCR, the reaction system contains primers that are specifically reverse-complementary paired with at least a portion of the sequence in the molecularly tagged adapter; Preferably, in the first round amplification step of the nested PCR, the reaction system further contains a targeted amplification reagent; Preferably, in the second round of amplification of the nested PCR, the reaction system contains primers that are specifically reverse complementary to at least part of the sequence in the molecularly tagged adapter; Preferably, in the first round amplification step of the nested PCR, the reaction system further contains a targeted amplification reagent.

9. The library construction method according to claim 6, wherein The step further includes a purification step, comprising performing magnetic bead purification on the second-round amplification product to obtain a purified product; Preferably, the method further comprises a library amplification step, comprising amplifying the purified product to obtain a library amplification product; Preferably, in the library amplification step, the reaction system contains at least one of a library amplification reagent, an I5 tag, and an I7 tag; Preferably, the method further comprises a purification step, comprising performing magnetic bead purification on the amplified product of the library to obtain a library that can be used for sequencing; Preferably, in the adapter ligation step, the nucleic acid sample is a sample that has undergone end repair and "A" addition reaction.

10. The library prepared by the library construction method according to any one of claims 6 to 9.

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