A method for co-detecting gene mutation, amplification and chromosome CNV with ultra-low starting DNA

Abstract

The present invention provides a method for co-detection of gene mutation, amplification and chromosome CNV of ultra-low starting amount DNA, which belongs to the field of gene detection technology, including: constructing a sequencing library for the detection target, the sequencing library includes an original template DNA fragment and a target region amplified fragment; the detection result is obtained by sequencing the sequencing library, the whole genome sequencing depth is 1X-3X, and the target region sequencing depth is ≥1000X. The technical solution provided by the present invention has extremely low requirements for the amount of starting DNA, and realizes the co-detection of gene mutation, amplification and chromosome CNV in the same library, greatly improving the detection sensitivity and detection application scope, reducing the operation steps, especially for special use scenarios, improving the labor intensity of operators.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a method for co-detecting ultra-low starting amount DNA gene mutation, amplification and chromosome CNV. Background Art

[0002] Gene mutation detection is an important field in modern medicine and biological research, and plays a vital role in the diagnosis, treatment and prevention of diseases. With the development of gene mutation detection technology, the detection of tumor mutation genes has been widely used in clinical practice, especially before targeted drugs are used to treat tumor patients, genetic testing must be performed first in accordance with domestic and international guidelines. For example, in non-small cell lung cancer, EGFR (epidermal growth factor receptor) gene mutations are closely related to the treatment options for cancer patients. Specific EGFR mutations, such as exon 19 deletions and p.L858R point mutations in exon 21, are associated with sensitivity to oral EGFR tyrosine kinase inhibitor (TKI) treatment. KRAS mutations are associated with low response rates to EGFR TKI treatment, and the detection of their mutation status helps predict patients' possible response to EGFR TKI treatment. In addition, KRAS mutations are also an indicator of poor prognosis. In adult diffuse gliomas, the detection of IDH1 / 2 and pTERT gene mutations has become an important criterion for molecular typing. Currently, commonly used gene detection methods include Sanger sequencing, digital titration polymerase chain reaction (ddPCR), real-time fluorescence quantitative PCR (qPCR) and high-throughput next-generation sequencing (NGS). Among them, Sanger sequencing has very high accuracy in base recognition and extremely low error rate, but it is expensive, has limited read length, and requires a large amount of DNA, generally 2 μg DNA. qPCR is low-cost, short-term, and compatible with multiple reactions to achieve the purpose of detecting multiple targets, but this method is heavily dependent on the specificity of primers and probes, and there is a certain amount of non-specific amplification. At the same time, this method cannot detect complex insertion and deletion mutations. ddPCR can detect extremely low-abundance target DNA, with very high sensitivity and accuracy, and can perform absolute quantification of target molecules, but this method also has some disadvantages, such as high cost, cumbersome operation, and limited throughput. None of the above three detection methods can obtain information on gene mutations and genomic CNVs at the same time. NGS technology introduces reversible termination ends to achieve sequencing while synthesis. A large amount of information on loci can be obtained, including information on gene mutations, gene fusions, and chromosomal variations (CNVs). To obtain mutation information in the target region, hybrid capture and amplicon library construction methods are currently commonly used. However, the hybrid capture library construction method requires the design of specific probes for the target region and requires a large amount of starting DNA, generally 100-200 ng DNA. The operation process is complicated, and many clinical samples cannot meet its requirements, causing many cancer patients to lose precious opportunities for targeted drug treatment.In addition, if it is necessary to obtain both gene mutation and chromosome variation information at the same time, a large number of probes need to be designed, which greatly increases the design difficulty and detection cost; although the amplicon library construction method can reduce the requirements for the amount of starting DNA, it can only obtain mutation information of a small amount of target regions and cannot obtain chromosome CNV information at the same time.

[0003] Common clinical sample types include tissue samples obtained through surgery or puncture, as well as liquid samples such as blood, pleural effusion, urine, and cerebrospinal fluid. Except for tissues obtained through surgery and some paraffin-embedded samples, it is difficult to obtain sufficient genomic DNA for molecular pathology testing in most samples. For example, puncture biopsy is one of the commonly used techniques for early diagnosis of tumors, and the volume of tissue obtained is about 7-9 mm. 3 / Article, it is difficult to obtain DNA for molecular testing from the remaining tissue after pathological examination. In order to maximize the use of the biopsy tissue to obtain a clear diagnosis of pathological types and molecular gene testing, a technology that requires low starting DNA amount and can simultaneously obtain multiple DNA variation information is urgently needed in clinical practice. Summary of the invention

[0004] To solve the above problems, the present invention provides a method for co-detection of gene mutation, amplification and chromosome CNV with ultra-low starting amount DNA. The technical solution provided by the present invention has extremely low requirements on the starting DNA amount, and realizes the co-detection of gene mutation, amplification and chromosome CNV in the same library, which greatly improves the detection sensitivity and detection scope, reduces the operation steps, and improves the labor intensity of operators, especially for special usage scenarios.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for co-detecting gene mutation, amplification and chromosome CNV of ultra-low starting DNA, comprising: constructing a sequencing library for the detection target, the sequencing library comprising an original template DNA fragment and a target region amplification fragment; the construction of the sequencing library comprises PCR amplification of the target region and amplification of the library, and the specific calculation method of the number of cycles of the PCR amplification is as follows: the sequencing depth of the target mutation site is ≥ 1000X; the number of target amplicon molecules in the library = 300mNλ 3 σ2 c+a ; Number of whole genome source molecules in the library = 3G × 300mλ 3 σ2 a / L; the number of target amplicon molecules in the library / the number of molecules from the whole genome in the library <1; λ∈[0.3, 0.5]; σ∈[0.5, 0.7]; in the above formula, 300 is the copy number of 1ng human genomic DNA, m is the initial DNA input, N is the number of target amplification regions, c is the number of cycles of target region amplification, λ is the recovery rate of magnetic bead purification, σ is the efficiency of adapter ligation, 3G is the number of bases in the human genome, 3*10 9 , L is the expected genome enzyme cutting length, and a is the number of cycles of library amplification; the detection result is obtained by sequencing the sequencing library, the whole genome sequencing depth is 1X-3X, and the target region sequencing depth is ≥1000X.

[0007] Furthermore, the method for constructing the sequencing library is as follows: 1) fragmenting the sample DNA; 2) performing PCR amplification on the target region of the DNA fragment; 3) purifying the amplified product, wherein the purified product contains the amplified fragment and the original template DNA molecule, and the product is end-repaired and A-added to obtain an A-added product; 4) connecting the A-added product to a sequencing adapter to form a library, and amplifying and purifying the library.

[0008] Furthermore, in the step 1), m≥1ng.

[0009] Furthermore, in the step 1), the sample DNA is fragmented by enzyme digestion.

[0010] Furthermore, the sequencing adapter connected in step 4) is a Y-shaped adapter with a molecular tag; the Y-shaped adapter has an index sequence in at least one chain in the non-complementary region.

[0011] Furthermore, in the step 4), the purification treatment is: selecting the purified library fragments of 480-500 bp by electrophoresis.

[0012] Furthermore, the sequencing library is sequenced to obtain the test results, including: quality control of the offline data; comparison of the quality-controlled data with the reference genome; indexing, and statistics of the comparison information of the target mutation site and calculation of the mutation frequency; deduplication of the bam file after indexing, and analysis of gene amplification and chromosome CNV.

[0013] The beneficial effects brought about by the technical solution provided by the present invention include:

[0014] 1. The present invention has extremely low requirements on the amount of starting DNA for library construction (as low as 1 ng), which can meet the requirements of most clinical sample types, including but not limited to tissue or liquid samples.

[0015] 2. The present invention can simultaneously detect gene mutation information in the target region, amplification information at the gene level and CNV information at the chromosome level, and the detection accuracy of gene mutation, gene amplification and chromosome CNV is high.

[0016] 3. The present invention is simple to operate, low in cost and high in flexibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The results of gene amplification detection of standard DNA with different starting amounts in Example 2 of the present invention are shown in the figure. The horizontal axis is the chromosome number and the vertical axis is the copy number.

[0019] Figure 2 This is a comparison of the detection results of the method of the present invention and the conventional low-depth WGS detection results for adult glioma samples in Example 5 of the present invention, where the horizontal axis is the chromosome number and the vertical axis is the copy number;

[0020] Figure 3 This is a comparison of the detection results of the method of the present invention and the conventional low-depth WGS detection results of two adult glioma samples in Example 5 of the present invention, where the horizontal axis is the chromosome number and the vertical axis is the copy number;

[0021] Figure 4 This is a comparison of the detection results of the method of the present invention and the conventional low-depth WGS detection results for another two adult glioma samples in Example 5 of the present invention. The horizontal axis is the chromosome number and the vertical axis is the copy number. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below by specific embodiments. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0023] As used herein, the words "comprises," "including," "having," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes listed elements is not necessarily limited to those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0025] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, immunology, laboratory science, gene sequencing technology, bioinformatics technology, and related fields.

[0026] Example 1

[0027] Material:

[0028] The samples were obtained from DNA extracted from the subject's tumor tissue.

[0029] method:

[0030] 1. DNA extraction and quantification

[0031] A nucleic acid extraction and purification kit (QIAamp DNA Mini Kit 250, QIAGEN) was used to extract and purify DNA. A DNA nucleic acid quantification kit (Qubit dsDNAHS Assay Kits, Thermo Fisher Scientific) was used to detect the concentration of the extracted DNA. The total amount of extracted DNA should be ≥1 ng.

[0032] 2. Sequencing library construction

[0033] 2.1 gDNA fragmentation

[0034] In order to reduce the loss of template DNA and ensure the enrichment of the target region under a small amount of subsequent DNA template, the DNA is sheared by enzyme fragmentation.

[0035] Prepare reaction system 1 on ice according to the following table:

[0036] Table 1. DNA fragmentation reaction system 1

[0037]

[0038] Note: Hieff smearase is a product of Yisheng Biotechnology (Shanghai) Co., Ltd.

[0039] After preparation, cap the tube tightly and vortex, then quickly centrifuge briefly and place on ice.

[0040] Reaction system 1 was reacted according to Table 2.

[0041] Table 2. DNA fragmentation reaction conditions

[0042]

[0043] 2.2 Targeted amplification of target regions

[0044] To further enrich the DNA molecules in the target region, PCR amplification was performed before connecting the library adapter. To avoid interactions between primers, the number of target regions was controlled within 50. This example selects important driver genes for early lung cancer lesions, which are also routine detection genes for lung cancer patients recommended in the NCCN clinical practice guidelines (EGFR, BRAF, KRAS) and important driver genes for adult gliomas (IDH1 / 2) to design mutation site detection primers. The base sequences of the primers are as follows:

[0045] Table 3. DNA amplification primer sequences

[0046]

[0047] Before the experiment, the DNA amplification primers were dissolved and diluted respectively, and then mixed to obtain mixed primer-F and mixed primer-R. The final mixed primer concentration was 10 μM.

[0048] Prepare the reaction system in a 0.2 ml PCR tube according to Table 4:

[0049] Table 4. Target region PCR amplification reaction system

[0050]

[0051] After preparation, cover the tube tightly and vortex, then centrifuge briefly and place in a PCR instrument to perform amplification reaction according to Table 5.

[0052] Table 5. PCR amplification reaction conditions

[0053]

[0054] Note: The PCR cycle number c needs to be estimated based on the sequencing amount and the starting input amount. The specific calculation method is as follows.

[0055] The number of PCR cycles in this reaction step has a great influence on the detection of the results. In the prior art, gene mutation detection requires a higher sequencing depth, such as 1000X or more, while chromosome CNV detection requires a lower sequencing depth, such as 1X-3X, and it is difficult to achieve the above functions through the same library. First, as the number of PCR cycles increases, DNA polymerase may encounter various challenges when replicating template DNA, such as damage to template DNA, nonspecific binding of primers, and mispairing of dNTPs. In each synthesis cycle, the polymerase may mistakenly insert a mismatched nucleotide into the new chain. Since PCR is an exponential growth process, even a small error rate will lead to a significant accumulation of incorrect bases after multiple cycles. On the contrary, too few PCR cycles will lead to insufficient number of templates available for detection, which will lead to failure of mutation detection at the target site; secondly, to ensure the accuracy of chromosome CNV detection and gene mutation and amplification detection results, it is necessary to ensure that the constructed library has high quality, especially the number of PCR amplification cycles in the target area and the number of library amplification cycles, which not only affects the accuracy of gene mutation, amplification and deletion detection, but also its amplification will affect the detection results of chromosome CNV, which increases the difficulty of constructing the same library. In order to solve the above problems, this application proposes a method for calculating the number of PCR amplification cycles for the target area, combined with subsequent electrophoresis to collect purified library fragments of a specific length, which can achieve ultra-low starting amount DNA gene mutation, amplification and chromosome CNV co-detection.

[0056] To ensure that the number of amplicon and whole genome reads in the offline data meets the analysis requirements, the number of amplicon molecules and the number of whole genome molecules in the final library should meet the following requirements:

[0057] Assumptions:

[0058] There is no bias in library amplification;

[0059] The sequencing process has no preference;

[0060] The efficiency of the enzyme during amplification is 100% and remains constant;

[0061] The recovery efficiency of magnetic bead purification remains consistent.

[0062] The sequencing depth of the target mutation site is ≥ 1000X;

[0063] Number of target amplicon molecules in the library = 300mNλ 3 σ2 c+a ;

[0064] The number of whole genome source molecules in the library = 3G × 300mλ 3 σ2 a / L;

[0065] The number of target amplicon molecules in the library / the number of whole genome source molecules in the library is less than 1;

[0066] λ∈[0.3, 0.5];

[0067] σ∈[0.5, 0.7];

[0068] In the above formula, 300 is the copy number of 1ng human genomic DNA, m is the initial DNA input, N is the number of target amplification regions, c is the number of cycles of target region amplification, λ is the magnetic bead purification recovery rate, σ is the adapter ligation efficiency, 3G is the number of bases in the human genome, 3*10 9 , L is the expected genome enzyme cutting length, and a is the number of cycles of library amplification.

[0069] If the initial DNA input amount m is 2 ng, the target amplification region N is 8, the number of subsequent library PCR amplification cycles a is 5, and the expected genome enzyme cutting length L is 200, c can be calculated from the above formula max ≤20. To ensure that the sequencing depth of the target mutation site is greater than 1000X, the number of target amplicon molecules in the library should be much greater than 1000, so 300mλ 3 σ2 c+a × p > 1000, p is the ratio of the library on the machine to the total library. If p is 5%, c min ≥4.

[0070] In actual operation, the cycle number c of the target region amplification ranges from: (c max +c min ) / 2 ±2.

[0071] 2.3 Purification of amplified products

[0072] Take the magnetic beads (Agencourt AMPure XP, Beckman Coulter, A63880) out of the refrigerator in advance, mix them, and leave them at room temperature for at least 30 minutes. Prepare enough fresh 80% ethanol solution (must be prepared and used immediately).

[0073] 1) After PCR, take out the PCR tube and centrifuge briefly, then add 75 μl (library volume: magnetic bead volume = 1:1.5) magnetic beads and vortex to mix (or mix with a pipette), let it stand at room temperature for 10 minutes, centrifuge it briefly and place it on a magnetic stand for 5 minutes. After the solution is clear, carefully aspirate the supernatant and discard it.

[0074] 2) Keep the sample on the magnetic rack, add 200 μl of freshly prepared 80% ethanol solution, let it stand for 30 seconds, then aspirate the solution and discard it.

[0075] 3) Repeat step 2), then remove as much ethanol as possible and keep the sample on the magnetic rack to dry, but the drying time should not exceed 3 minutes.

[0076] 4) Add 51 μl of nuclease-free water for elution, vortex and centrifuge, let stand at room temperature for 10 minutes, then place on a magnetic stand for about 5 minutes, and after the solution is clarified, pipette 50 μl of supernatant into a new PCR tube. The collected supernatant contains the amplified fragments of the target region and the template DNA fragments, which can be used for subsequent library construction.

[0077] 2.4 Add A to the end

[0078] Prepare the reaction system on ice according to Table 6:

[0079] Table 6. End-filling and A-addition reaction system

[0080]

[0081] After thorough mixing, place on a PCR instrument and perform the reaction according to Table 7.

[0082] Table 7. End-filling and A addition reaction conditions

[0083]

[0084] 2.5 Connector connection

[0085] After the above reaction is completed, the reaction system 4 is prepared according to Table 8, and after thorough mixing, the mixture is centrifuged and placed in a PCR instrument to react according to Table 9.

[0086] Table 8. Adapter ligation reaction system 4

[0087]

[0088] Table 9. Adapter ligation reaction conditions

[0089]

[0090] 2.6 Purification of ligation products

[0091] Take the magnetic beads (Agencourt AMPure XP, Beckman Coulter, A63880) out of the refrigerator in advance, mix them, and leave them at room temperature for at least 30 minutes. Prepare enough fresh 80% ethanol solution (must be prepared and used immediately).

[0092] 1) After the ligation reaction is completed, take out the PCR tube and centrifuge briefly, then add 93.5μl (library volume: magnetic bead volume = 1:1) magnetic beads and vortex to mix (or mix with a pipette), let it stand at room temperature for 10 minutes, centrifuge it briefly, and place it on a magnetic stand for 5 minutes. After the solution is clear, carefully aspirate the supernatant and discard it.

[0093] 2) Keep the sample on the magnetic rack, add 200 μl of freshly prepared 80% ethanol solution, let it stand for 30 seconds, then aspirate the solution and discard it.

[0094] 3) Repeat step 2), then remove as much ethanol as possible and keep the sample on the magnetic rack to dry, but the drying time should not exceed 3 minutes.

[0095] 4) Add 25 μl of nuclease-free water for elution, vortex to mix, centrifuge briefly, let stand at room temperature for 10 min, then place on a magnetic rack for about 5 min. After the solution is clarified, pipette 23 μl of supernatant into a new PCR tube.

[0096] 2.7 Library Amplification

[0097] Prepare the reaction system according to Table 10

[0098] Table 10. Library amplification reaction system

[0099]

[0100] After the prepared reaction system is fully mixed, place it on a PCR instrument and react according to Table 11.

[0101] Table 11. Library amplification reaction conditions

[0102]

[0103] 2.8 Library purification

[0104] 1) After the PCR reaction is completed, take out the PCR tube and centrifuge briefly, then add 50μl (library volume: magnetic bead volume = 1:1) magnetic beads and vortex to mix (or mix with a pipette), let it stand at room temperature for 10 minutes, centrifuge it briefly, and place it on a magnetic stand for 5 minutes. After the solution is clear, carefully aspirate the supernatant and discard it.

[0105] 2) Keep the sample on the magnetic rack, add 200 μl of freshly prepared 80% ethanol solution, let it stand for 30 seconds, then aspirate the solution and discard it.

[0106] 3) Repeat step 2), then remove as much ethanol as possible and keep the sample on the magnetic rack to dry, but the drying time should not exceed 3 minutes.

[0107] 4) Add 30 μl of nuclease-free water for elution, vortex to mix, centrifuge briefly, let stand at room temperature for 10 minutes, then place on a magnetic stand for about 5 minutes. After the solution is clarified, aspirate the supernatant into a new PCR tube.

[0108] 5) The library concentration should be greater than 0.1 ng / μl, and the purified library fragments of 480-500 bp should be collected by electrophoresis. If the library quality does not meet the requirements, the library should be reconstructed.

[0109] 3. Sequencing

[0110] According to the sequencer instructions, use Illumina or BGI sequencer for sequencing, and the sequencing data volume is 4-10G.

[0111] 4. Results Analysis

[0112] The data were processed using trimmomatic and fastp software to remove the adapter sequences and filter the low-quality sequences. The processed data were then aligned with the human genome reference sequence hg19 using the BWA MEM algorithm. The samtools were used to create an index after the aligned bam file was obtained. The mpileup command was used to count the alignment information of the target mutation site and calculate the mutation frequency. The indexed bam file was deduplicated, and the ichorCNV software was used for gene amplification and chromosome CNV analysis.

[0113] Example 2: Mutation testing of standards

[0114] The pan-tumor 800 gDNA standard (Cat. No.: GW-OGTM800) of Shenzhen Jingliang Technology Co., Ltd. was used as a test sample for the detection of the method of the present invention. The frequencies of the mutant genes involved in the standard are shown in Table 12:

[0115] Table 12. Frequency of gene mutations detected in the standard products

[0116]

[0117] 10 ng, 5 ng and 2 ng of the standard were taken as materials for detection according to the method of Example 1. Compared with the mutation frequency of the target site in the standard, the results of the detection by the method of the present invention are basically consistent with those of the standard, and the specific results are shown in Table 13. At the same time, the results of the gene amplification detection by the method of the present invention are also consistent with those of the standard, and the specific results are shown in Tables 14 and Figure 1 .

[0118] Table 13. Comparison of mutation detection results of the present invention with those of standard products

[0119]

[0120] Table 14. Comparison of gene amplification results of the present invention with those of standard products

[0121]

[0122] Example 3: Comparison of mutation detection results at different PCR cycle numbers

[0123] In order to compare the effects of different PCR cycle numbers on mutation detection, 2 ng of Jingliang Pan-Tumor 800 standard was used for detection by this method, and the PCR cycle numbers were set to 5, 13, and 20, respectively. The mutation detection results of different cycle numbers are shown in Table 15. The results show that when the PCR cycle number is too large, the accumulation of incorrect bases at the mutation site increases significantly, such as the number of C and G bases at the BRAF p.V600E site increases significantly, while too few PCR cycles lead to insufficient coverage of mutation site reads, affecting the calculation of mutation frequency, such as the total coverage of the BRAF p.V600E site is only 165X, of which there are only 9 mutation reads.

[0124] Table 15. Mutation detection results at different cycle numbers

[0125]

[0126] Among them, ref represents the reference base, mut represents the mutant base, and VAF represents the mutation frequency calculated by this method.

[0127] Example 4: Detection of actual clinical samples of lung adenocarcinoma

[0128] In order to verify the accuracy of the method of the present invention for gene mutation detection, 10 ng of lung adenocarcinoma tumor tissue DNA was taken for detection by the method of the present invention, and more than 200 ng of corresponding gDNA was taken for detection by Onco Focus lung cancer selected panel (Beijing PanGenomics Medical Testing Laboratory Co., Ltd.). The specific results of mutation detection are shown in Table 16, and the results show that the two are basically consistent.

[0129] Table 16. Comparison of mutation detection results in clinical samples of lung adenocarcinoma

[0130]

[0131] Example 5: Detection of actual clinical samples of adult glioma

[0132] To further illustrate the wide applicability of this method, 10 ng of adult glioma tumor tissue gDNA was used for the detection of this method, and 100 ng of the corresponding gDNA was used for brain six-item panel detection (Beijing PanGenomics Medical Laboratory Co., Ltd.) and WGS (2X) detection. The specific test results are shown in Tables 17-18, and the chromosome CNV test results are shown in Figure 2-4 .

[0133] Table 17. Comparison of mutation detection results in adult gliomas

[0134]

[0135] Table 18. Comparison of CNV detection results in adult glioma samples

[0136]

[0137] The numbers in the table represent chromosome numbers, p represents the long arm, q represents the short arm, + represents amplification, and - represents deletion.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for co-detection of ultra-low starting DNA gene mutation, amplification and chromosome CNV, wherein the co-detection method is for non-disease diagnosis and treatment purposes, characterized in that: include: Constructing a sequencing library for the detection target, wherein the sequencing library includes original template DNA fragments and target region amplified fragments; The construction of the sequencing library includes PCR amplification of the target region and amplification of the library. The specific calculation method of the number of cycles of the PCR amplification is as follows: The sequencing depth of the target mutation site is ≥1000X; Number of target amplicon molecules in the library = 300 mNλ 3 σ2 c+a ; The number of whole genome source molecules in the library = 3G × 300mλ 3 σ2 a / L; The number of target amplicon molecules in the library / the number of whole genome source molecules in the library is less than 1; λ∈[0.3, 0.5]; σ∈[0.5, 0.7]; In the above formula, 300 is the copy number of 1ng human genomic DNA, m is the initial DNA input, N is the number of target amplification regions, c is the number of cycles of target region amplification, λ is the magnetic bead purification recovery rate, σ is the adapter ligation efficiency, and 3G is the number of bases in the human genome 3*10 9 , L is the expected genome enzyme cutting length, a is the cycle number of library amplification; The detection results are obtained by sequencing the sequencing library, the whole genome sequencing depth is 1X-3X, and the target region sequencing depth is ≥1000X.

2. The co-detection method according to claim 1, characterized in that: The method for constructing the sequencing library is as follows: 1) Sample DNA fragmentation processing; 2) PCR amplification of the target region of the DNA fragment; 3) Purifying the amplified product, the purified product contains the amplified fragment and the original template DNA molecule, and the product is end-repaired and A-added to obtain an A-added product; 4) Connecting the A-added products to sequencing adapters to form a library, and amplifying and purifying the library.

3. The co-detection method according to claim 2, characterized in that: In the step 1), m≥1ng.

4. The co-detection method according to claim 2, characterized in that: In the step 1), the sample DNA is fragmented by enzyme digestion.

5. The co-detection method according to claim 2, characterized in that: The sequencing adapter connected in step 4) is a Y-shaped adapter with a molecular tag; the Y-shaped adapter has an index sequence in at least one chain in the non-complementary region.

6. The co-detection method according to claim 2, characterized in that: In the step 4), the purification process is to select the purified library fragments of 480-500 bp by electrophoresis.

7. The co-detection method according to claim 1, characterized in that: Sequencing the sequencing library to obtain a detection result includes: Quality control of offline data; The quality-controlled data are compared with the reference genome; Establish an index, collect statistics on the comparison information of the target mutation site, and calculate the mutation frequency; The indexed bam files were deduplicated and analyzed for gene amplification and chromosome CNV.

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