Method for detecting gene copy number variation based on amplicon sequencing technology

Abstract

The invention discloses a method for detecting gene copy number variation based on an amplicon sequencing technology, which can accurately calculate the input molecule number of a target area based on the amplicon sequencing technology by introducing a correction template into a PCR amplification reaction system for correcting the amplification efficiency and amplification preference of a to-be-detected sample. The variation condition of the copy number of the target area of the to-be-detected sample can be accurately judged according to the comparison condition of the copy number of the to-be-detected sample and the number of the input molecules of the internal reference area.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a method for detecting gene copy number variation based on amplicon sequencing technology. Background Art

[0002] Amplicon sequencing is a technology used to amplify and sequence specific nucleotide sequences at high throughput. It is widely used to study the variation, mutation or expression level of specific genes or genomic regions.

[0003] Copy Number Variation (CNV) refers to the loss or gain of a DNA sequence of a certain size (generally tens of bp to several Mp) compared to the reference genome. In the method of detecting gene copy number variation based on amplicon sequencing technology, specific primers are first designed for the target region, and the target detection region is amplified into a large number of replicas, i.e., amplicons, through PCR amplification reaction, and then these amplicons are enriched using the index sequence carried by the primers, and finally the enriched amplicons are subjected to high-throughput sequencing to obtain the corresponding information.

[0004] The advantages of the above methods include: (1) the ability to selectively amplify and sequence genes or genomic regions of interest, with flexible design to meet different needs; (2) high sensitivity, capable of detecting low copy number variants or mutations; (3) high throughput, capable of simultaneously amplifying and sequencing multiple samples to be tested, improving efficiency; (4) relatively low cost compared to whole genome or whole transcriptome sequencing. However, the library construction method based on PCR amplification reaction has significant differences in the results obtained by sequencing different batches of experiments within a sample and between samples, due to the instability of the PCR amplification reaction that affects the quality of the amplified product, thereby affecting the accuracy and stability of gene copy number detection and leading to frequent false positive results. Summary of the invention

[0005] In view of the existing technical problems, the present application provides a method for detecting gene copy number variation based on amplicon sequencing technology.

[0006] The method described in this application comprises the following steps:

[0007] (1) constructing a PCR amplification reaction system, the PCR amplification reaction system comprising: a test sample DNA, a first calibration template and a second calibration template designed for a target region and an internal reference region in a reference genome sequence, respectively, a first primer for specifically amplifying the target region in the test sample DNA and the first calibration template, and a second primer for specifically amplifying the internal reference region in the test sample DNA and the second calibration template;

[0008] (2) performing a PCR amplification reaction to obtain an amplicon library;

[0009] (3) sequencing the amplicon library to obtain a sequencing library, and grouping the sequences in the sequencing library according to their characteristics to obtain a target region product group, an internal reference region product group, a first correction template product group, and a second correction template product group;

[0010] (4) calculating the average amplification efficiency K1 of the first correction template in the PCR amplification reaction according to the sequencing depth of the first correction template product group, and calculating the average amplification efficiency K2 of the second correction template in the PCR amplification reaction according to the sequencing depth of the second correction template product group;

[0011] (5) obtaining the number of molecules A0 of the target region input in the PCR amplification reaction by the ratio of the number of molecules A of the target region product group to K1, and obtaining the number of molecules B0 of the internal reference region input in the PCR amplification reaction by the ratio of the number of molecules B of the internal reference region product group to K2;

[0012] (6) judging the copy number variation of the target region in the DNA of the sample to be tested according to the ratio of A0 to B0;

[0013] The design and synthesis of the first calibration template and the second calibration template are based on the same sequence of the respective reference regions, and the two parts of the sequence in the middle of the respective reference regions are respectively selected and replaced with the general formula N in the synthesis process. x Sequence, where N x Represents a sequence consisting of x random bases N, where x is 3 to 5.

[0014] Several optional methods are also provided below, but they are not intended to be additional limitations on the above-mentioned overall solution, but are merely further supplements or preferences. Under the premise that there are no technical or logical contradictions, each optional method can be combined with the above-mentioned overall solution separately, and multiple optional methods can also be combined.

[0015] Optionally, the concentrations of the first calibration template and the second calibration template in the PCR amplification reaction system are both 0.2-0.4 fM.

[0016] Optionally, during sequencing, two parts of sequence N x as an identification sequence for each calibration template; or,

[0017] The two parts of the sequence N x The sequence between is replaced by an ID sequence, wherein the ID sequence consists of 5 to 7 fixed bases and is used as an identification sequence for each correction template during the sequencing process.

[0018] Optionally, the N xIt is set at an interval with the ID sequence, and the interval length is 3 to 5 bases.

[0019] Optionally, the sequences in the first correction template and the second correction template are both single-stranded.

[0020] Optionally, there are multiple target regions, the sequence length of each target region is 100 to 150 bp, and multiple first correction templates are provided accordingly.

[0021] Optionally, the sequence of the reference region is compared, and each correction template product group is clustered according to different combination sequences of random bases N to obtain a series of sequencing depths (Reads count) of different sequences and determine the sequence cluster threshold. All sequences are divided into high-depth clusters and low-depth clusters according to the sequence cluster threshold, and the low-depth clusters are eliminated. The average number of clusters of all high-depth clusters is calculated, that is, the K1 and K2 are obtained respectively.

[0022] Optionally, the low-depth cluster is a sequence cluster whose sequencing depth is less than a sequence cluster threshold d, wherein the sequence cluster threshold d is the square root of the average value of all sequencing depths in the remaining clusters after removing sequence clusters with less than 2 molecules.

[0023] Optionally, there are multiple internal reference regions, the sequence length of each internal reference region is 100 to 150 bp, and multiple second correction templates are set corresponding to all internal reference regions.

[0024] Optionally, the copy number of the internal reference region is 2.

[0025] Optionally, in step (5), multiple B0 are obtained and the average number of input molecules of all internal reference region product groups is calculated. In step (6), A0 and ratio.

[0026] Optionally, the PCR amplification reaction system further comprises a specific amplification reaction solution, a sequencing adapter primer and an enrichment amplification reaction solution;

[0027] The step (2) comprises:

[0028] Performing a first round of amplification reaction using primers, a specific amplification reaction solution, a sample DNA to be tested, and each calibration template, and obtaining a first amplification product mixture after purification;

[0029] Performing a second round of amplification reaction using the sequencing adapter primer, the enriched amplification reaction solution and the first amplification product mixture to obtain a second amplification product mixture;

[0030] The second amplification product mixture is purified to obtain the sequencing library.

[0031] Compared with the prior art, the present application introduces a correction template into the PCR amplification reaction system to correct the amplification efficiency and amplification preference in the sample to be tested, thereby being able to accurately calculate the number of input molecules in the target detection area based on amplicon sequencing technology, and accurately determine the variation in the copy number of the target area of ​​the sample to be tested based on the comparison with the number of input molecules in the internal reference area. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart of a method for detecting gene copy number variation based on amplicon sequencing technology in one embodiment of the present application. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] See also Figure 1 An embodiment of the present application provides a method for detecting gene copy number variation based on amplicon sequencing technology, comprising the following steps:

[0036] (1) constructing a PCR amplification reaction system, the PCR amplification reaction system comprising: a test sample DNA, a first calibration template and a second calibration template designed for the target region and the internal reference region in the reference genome sequence, respectively, a first primer for specifically amplifying the target region and the first calibration template in the test sample DNA, and a second primer for specifically amplifying the internal reference region and the second calibration template in the test sample DNA;

[0037] (2) performing a PCR amplification reaction to obtain an amplicon library;

[0038] (3) sequencing the amplicon library to obtain a sequencing library, and grouping the sequencing results in the sequencing library according to the characteristics of each sequence to obtain a target region product group, an internal reference region product group, a first correction template product group, and a second correction template product group;

[0039] (4) calculating the average amplification efficiency K1 of the first calibration template in the PCR amplification reaction according to the sequencing depth of the first calibration template product group, and calculating the average amplification efficiency K2 of the second calibration template in the PCR amplification reaction according to the sequencing depth of the second calibration template product group;

[0040] (5) The number of molecules A0 input into the detection region in the PCR amplification reaction is obtained by the ratio of the number of molecules A of the target region product group to K1, and the number of molecules B0 input into the internal reference region in the PCR amplification reaction is obtained by the ratio of the number of molecules B of the internal reference region product group to K2;

[0041] (6) According to the ratio of A0 and B0, the copy number of the target region in the DNA of the sample to be tested is determined.

[0042] Among them, the design and synthesis of the first correction template and the second correction template are based on the same sequence of the respective reference regions, and the two parts of the sequence in the middle of the respective reference regions are respectively selected and replaced with the general formula N in the synthesis process. x Sequence, where N x Represents a sequence consisting of x random bases N, where x is 3 to 5.

[0043] In order to achieve effective grouping, two parts of sequence N are used in the sequencing process. x As the identification sequence of each calibration template; or, the two-part sequence N x The sequence between them is replaced by an ID sequence, which is composed of 5 to 7 fixed bases and is used as an identification sequence for each correction template during the sequencing process, for example, CGATGT.

[0044] In the present application, the sequences in the first calibration template and the second calibration template are both single-stranded. The random base N is A, T, C or G, the first calibration template matches the target region sequence at not less than 99%, and the second calibration template matches the internal reference region at not less than 99%. It is theoretically believed that in the same amplification reaction system, the target region in the sample DNA to be tested has a similar amplification efficiency as the first calibration template, and the internal reference region has a similar amplification efficiency as the second calibration template. The number of molecules refers to the sequencing depth (Reads Count) obtained by sequencing each sequence region and comparing it with the reference sequence.

[0045] Specifically, N in each correction template x The interval between the ID sequence and the ID sequence is 3 to 5 bases. x It is N5, and the interval length with the ID sequence is 4 bases, and these 4 bases are consistent with the bases of the reference sequence.

[0046] In one embodiment, there are multiple target regions, each of which is 100-150 bp long, and multiple first calibration templates are provided accordingly. For different target regions and first calibration templates, multiple groups of first primers are provided accordingly.

[0047] The copy number of the internal reference region is 2, and the internal reference region is, for example, selected from at least one of GAPDH, β-actin, 18S rRNA, 28S rRNA, B2M, HPRT, TBP, RPPH1 and PRR30.

[0048] In step (1), the concentrations of the first calibration template and the second calibration template in the PCR amplification reaction system are both 0.2 to 0.4 fM. 2x For example, when there are 10 random bases in the correction template, theoretically 4 10 In the PCR amplification reaction system, when the number of each correction template used is small enough, the number of each combination is close to 1 in theory, that is, from 4 10 About a hundred combinations are randomly selected from the combinations, for example, about 100 to 200 combinations are randomly selected.

[0049] In step (4), the sequence of the reference region is compared, and each correction template generation group is clustered according to the different sequence conditions of N, to obtain a series of sequencing depths of different sequences and determine the sequence cluster threshold, and all sequences are divided into high-depth clusters and low-depth clusters according to the sequence threshold, and the low-depth clusters are eliminated, and the average values ​​of all high-depth clusters are calculated, that is, the K1 and K2 are obtained respectively. Among them, the low-depth cluster is a sequence with a sequencing depth less than the sequence cluster threshold d, and the sequence cluster threshold d is the square root of the average value of all sequencing depths in the remaining clusters after eliminating sequence clusters with less than 2 molecules.

[0050] Furthermore, there are multiple internal reference regions, each of which has a sequence length of 100 to 150 bp, and multiple second calibration templates and multiple sets of second primers are provided for all internal reference regions. Accordingly, in step (5), multiple B0s are obtained and the average number of input molecules of all internal reference region product groups is calculated. In step (6), A0 and ratio.

[0051] In one embodiment, the PCR amplification reaction system further includes a specific amplification reaction solution, a sequencing adapter primer and an enrichment amplification reaction solution.

[0052] In one embodiment, step (2) includes: performing a first round of amplification reaction using primers, a specific amplification reaction solution, a sample DNA to be tested, and calibration templates to obtain a first amplification product mixture; performing a second amplification reaction using sequencing adapter primers, an enrichment amplification reaction solution, and the first amplification product mixture to obtain a second amplification product mixture; and purifying the second amplification product mixture to obtain a sequencing library.

[0053] Further, each primer is pre-mixed into a primer mixture, and when used, the final concentration of each primer in the amplification reaction system is 0.02-0.03 μM, preferably 0.025 μM. Each calibration template is pre-mixed into a calibration template mixture, and when used, the final concentration of each calibration template in the amplification reaction system is 0.2-0.4 fM, preferably 0.3 fM.

[0054] In the present application, by adopting specific amplification reaction solution and enrichment amplification reaction solution, and combining PCR amplification reaction optimization, the stability of PCR amplification can be guaranteed, especially when applied to multiplex PCR to detect copy number variations in multiple regions (genes), the stability and efficiency of amplification can be effectively guaranteed.

[0055] The technical solution of this application is further explained below in combination with specific application scenarios and experimental parameters.

[0056] Application Example 1: Detection of SMA gene copy number variation

[0057] This application example is aimed at SMA gene screening, and the detection of EXON7 and EXON8 in SMN1 is used to determine the variation of the SMA gene copy number. The sequences involved in this example are shown in Table 1:

[0058] Table 1 Sequence list involved in Application Example 1

[0059]

[0060]

[0061]

[0062] Steps:

[0063] 1. Prepare the DNA sample to be tested

[0064] Extract human peripheral blood genomic DNA according to the instructions of nucleic acid extraction or purification reagents. The recommended DNA detection usage range is: 2ng≤DNA concentration≤4ng, purity: 260 / 280>1.8, 260 / 230>1.6.

[0065] 2. Construct the first round of amplification reaction system

[0066] According to the system in Table 2, the specific amplification reaction solution, primer mixture and correction template mixture were thawed on ice respectively. After thawing, each solution was vortexed for 3-5 seconds to mix and centrifuged for standby use; 15 μL of the reaction mixture was dispensed into each well of the PCR plate, and then 5 μL of the sample DNA to be tested was added to each reaction well. After sealing, vortexed and mixed, and centrifuged instantly to expel the bubbles at the bottom of the tube, and the droplets on the tube wall and the sealing film were collected at the bottom of the tube.

[0067] Table 2 Amount of each component in the amplification reaction system

[0068]

[0069] The specific amplification reaction solution was Equinox Multiplex PCR Master Mix (Watchmaker Genomics); the final concentrations of P1 to P4 in the primer mixture in the amplification reaction system were 0.025 μM respectively; P1 could amplify two regions in the SMN1 gene EXON7 and the SMN2 gene EXON7, and P2 could amplify two regions in the SMN1 gene EXON8 and the SMN2 gene EXON8. The final concentrations of Q1, Q2, Q3, and Q4 in the correction template mixture in the amplification reaction system were 0.3 fM.

[0070] 3. Perform the first round of amplification reaction

[0071] The first round of amplification reaction was carried out according to Table 3. After amplification, each well of the PCR plate was filled up to 50 μL with water and then purified by magnetic beads to obtain a purified first amplification product mixture.

[0072] Table 3 First round amplification reaction conditions

[0073]

[0074]

[0075] 4. Construct the second round of amplification reaction system

[0076] According to Table 4, the enrichment amplification reaction solution and sequencing adapter primer set were thawed on ice, vortexed for 3-5 seconds to mix, centrifuged instantly, and placed on ice for use; a new PCR plate was taken, and 25 μL of the enrichment amplification reaction solution was dispensed into each well of the PCR plate; the corresponding enrichment adapter primer set was added to each reaction well according to the required index position, and 23 μL of the first round of library product after purification was added, and the PCR plate was sealed with a sealing film, vortexed for 5-10 seconds, and centrifuged instantly to expel the bubbles at the bottom of the tube, and the droplets on the tube wall and the sealing film were collected at the bottom of the tube.

[0077] Table 4 Second round amplification reaction system

[0078]

[0079] The enrichment amplification reaction solution was Equinox Library Amplification Kit (Watchmaker Genomics).

[0080] 5. Perform the second round of amplification reaction

[0081] The second round of amplification reaction was carried out according to Table 5, and magnetic bead purification was performed after amplification to obtain a purified second amplification product mixture as the amplification product mixture for sequencing.

[0082] Table 5 Second round amplification reaction conditions

[0083]

[0084] 6. Sequence the library on the Illumina sequencing platform.

[0085] 7. Analyze the sequencing results:

[0086] (1) Perform quality processing on the data after it is downloaded from the machine, remove the adapter sequence, and perform data quality processing (clean fastq);

[0087] (2) The data were grouped according to the characteristics of each sequence and compared with the genomic reference sequence after pre-shielding the SMN2 region. The sequencing depth of regions EXON7, EXON8 and each internal reference region in the SMN1 gene was obtained, i.e., read count, based on the base site differences between SMN1 and SMN2.

[0088] (3) calculating the average amplification efficiency K1 of Q1 in the PCR amplification reaction according to the sequencing depth of Q1, calculating the average amplification efficiency K1 of Q2 in the PCR amplification reaction according to the sequencing depth of Q2, calculating the average amplification efficiency K2 of Q3 in the PCR amplification reaction according to the sequencing depth of Q3, and calculating the average amplification efficiency K2 of Q4 in the PCR amplification reaction according to the sequencing depth of Q4;

[0089] (4) The number of molecules A0 of EXON7 input in the PCR amplification reaction is obtained by the ratio of the number of molecules A (Reads count) of EXON7 to K1 of Q1, and the number of molecules A0 of EXON8 input in the PCR amplification reaction is obtained by the ratio of the number of molecules A (Reads count) of EXON8 to K1 of Q2; the number of molecules B0 of the internal reference region RPPH1 input in the PCR amplification reaction is obtained by the ratio of the number of molecules A of the internal reference region RPPH1 to K2 of Q3, and the number of molecules B0 of the internal reference region PRR30 input in the PCR amplification reaction is obtained by the ratio of the number of molecules A of the internal reference region PRR30 to K2 of Q4, and the result is obtained.

[0090] The average number of molecules invested in the internal reference region RPPH1 and the internal reference region PRR30

[0091] (5) Calculate the number of molecules invested A0 and the average number of molecules invested in each target area According to the ratio, the variation of the copy number of the EXON7 and EXON8 regions in the SMN1 gene of the sample DNA to be tested is determined;

[0092] Results and Analysis

[0093] In this application example, the DNA samples to be tested are sample 1 and sample 2, and the test results are shown in Table 6. Sample 1 and sample 2 were verified by the gold standard method multiple ligation-dependent probe amplification (MLPA), wherein, after MLPA verification, it was known that sample 1 was a normal sample with 2 copies of the SMN1 gene, and sample 2 was a deletion type sample with 1 copy of the SMN1 gene. In this application, the difference in base sites can be combined to shield the interference of the homologous sequence SMN2 and achieve accurate analysis of the SMN1 gene.

[0094] Table 6 Test results of application example 1

[0095]

[0096] Application Example 2DMD Detection

[0097] This application example is aimed at DMD gene screening, and detects the variation of the copy number of the intron-Exon 45 and Exon 46 regions in DMD. The sequences involved in this example are shown in Table 7.

[0098] Table 7 Sequence list involved in Application Example 2

[0099]

[0100]

[0101] The specific operation steps refer to the experimental steps of Application Example 1. In this application example, the sample DNA to be tested is sample 3, and the test results are shown in Table 8, and are verified by digital PCR. Among them, digital PCR verification shows that sample 3 to be tested is a heterozygous deletion of DMD exon 45-46.

[0102] Table 8 Test results of application example 2

[0103]

[0104]

[0105] Application Example 3

[0106] This application example is aimed at DMD gene screening, detecting the variation of the copy number of the intron-Exon 45 and Exon 46 regions in DMD. The sequences involved in this example are shown in Table 9:

[0107] Table 9 Sequence list involved in Application Example 3

[0108]

[0109]

[0110] The specific operation steps refer to the experimental steps of Application Example 1. In this application example, the sample DNA to be tested is sample 3, and the test results are shown in Table 10, and are verified by digital PCR. Among them, digital PCR verification shows that sample 3 is a heterozygous deletion of DMD exon 45-46.

[0111] Table 10 Test results of application example 3

[0112]

[0113] Application Example 4

[0114] The amplicon capture panel in this application example contains 156 amplicon primer coverage areas (i.e., primer mixtures), including specific amplification primers for EXON7 and EXON8 of the SMN1 gene (see Table 1) and specific amplification primers for intron-Exon 45 and Exon 46 of the DMD gene (see Table 7). In addition to detecting the copy number changes of the SMN1 gene and DMD gene in the sample, it can also detect point mutations of genes such as HBB, GJB2, SLC25A13, and SLC26A4. In this application example, the same batch of experiments were performed on samples 1, 2, and 3 to be tested. The specific operation steps refer to Application Example 1, and the test results are shown in Table 11.

[0115] Table 11 Test results of application example 4

[0116]

[0117] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A method for detecting gene copy number variation based on amplicon sequencing technology, characterized in that: The following steps are involved: (1) constructing a PCR amplification reaction system, the PCR amplification reaction system comprising: a test sample DNA, a first calibration template and a second calibration template designed for a target region and an internal reference region in a reference genome sequence, respectively, a first primer for specifically amplifying the target region in the test sample DNA and the first calibration template, and a second primer for specifically amplifying the internal reference region in the test sample DNA and the second calibration template; (2) performing a PCR amplification reaction to obtain an amplicon library; (3) sequencing the amplicon library to obtain a sequencing library, and grouping the sequences in the sequencing library according to their characteristics to obtain a target region product group, an internal reference region product group, a first correction template product group, and a second correction template product group; (4) calculating the average amplification efficiency K1 of the first correction template in the PCR amplification reaction according to the sequencing depth of the first correction template product group, and calculating the average amplification efficiency K2 of the second correction template in the PCR amplification reaction according to the sequencing depth of the second correction template product group; (5) obtaining the number of molecules A0 input into the detection region in the PCR amplification reaction by the ratio of the number of molecules A of the target region product group to K1, and obtaining the number of molecules B0 input into the internal reference region in the PCR amplification reaction by the ratio of the number of molecules B of the internal reference region product group to K2; (6) Determining the copy number of the target region in the DNA of the sample to be tested based on the ratio of A0 to B0; The design and synthesis of the first calibration template and the second calibration template are based on the same sequence of the respective reference regions, and the two parts of the sequence in the middle of the respective reference regions are respectively selected and replaced with the general formula N in the synthesis process. x Sequence, where N x Represents a sequence consisting of x random bases N, where x is 3 to 5.

2. The method according to claim 1, characterized in that The concentrations of the first calibration template and the second calibration template in the PCR amplification reaction system are both 0.2-0.4 fM.

3. The method according to claim 1, characterized in that In the sequencing process, two parts of sequence N x as an identification sequence for each calibration template; or, The two parts of the sequence N x The sequence between is replaced by an ID sequence, wherein the ID sequence consists of 5 to 7 fixed bases and is used as an identification sequence for each correction template during the sequencing process.

4. The method according to claim 1, characterized in that The sequences in the first calibration template and the second calibration template are both single-stranded.

5. The method according to claim 1, characterized in that In step (4), the sequence of the reference region is compared, and each correction template product group is clustered according to different combination sequences of random bases N to obtain a series of sequencing depths of different sequences and determine the sequence cluster threshold. All sequences are divided into high-depth clusters and low-depth clusters according to the sequence cluster threshold, and the low-depth clusters are eliminated. The average number of clusters of all high-depth clusters is calculated, that is, K1 and K2 are obtained respectively.

6. The method according to claim 5, characterized in that The low-depth cluster is a sequence cluster whose sequencing depth is less than a sequence cluster threshold d, where d is the square root of the average of all sequencing depths in the remaining clusters after removing sequence clusters with less than 2 molecules.

7. The method according to claim 1, characterized in that There are multiple target sequences, the sequence length of each target region is 100 to 150 bp, and multiple first correction templates are set corresponding to all target regions.

8. The method according to claim 1, characterized in that: There are multiple internal reference regions, the sequence length of each internal reference region is 100 to 150 bp, and multiple second correction templates are set corresponding to all internal reference regions; The copy number of the internal reference region is 2.

9. The method according to claim 8, characterized in that In step (5), multiple B0 are obtained and the average number of input molecules of all internal reference area product groups is calculated. In step (6), A0 and ratio.

10. The method according to claim 1, characterized in that The PCR amplification reaction system also includes a specific amplification reaction solution, a sequencing adapter primer and an enrichment amplification reaction solution; Step (2) comprises: Performing a first round of amplification reaction using primers, a specific amplification reaction solution, a sample DNA to be tested, and each calibration template, and obtaining a first amplification product mixture after purification; Performing a second round of amplification reaction using the sequencing adapter primer, the enriched amplification reaction solution and the first amplification product mixture to obtain a second amplification product mixture; The second amplification product mixture is purified to obtain the amplicon library.

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