Method and kit for constructing multi-gene and multi-mutation sequencing libraries for targeted treatment of lung cancer

Abstract

The present invention relates to a method and kit for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer, and belongs to the field of gene sequencing technology. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer comprises the following steps: designing a multiple PCR amplification primer set based on a targeted diagnostic gene, obtaining a first round of PCR amplification products through a first round of PCR amplification; adding a magnetic bead liquid to the first round of PCR amplification products, purifying the magnetic beads to obtain a purified product I with magnetic beads, and performing enzyme digestion; adding a magnetic bead liquid to the purified product I with magnetic beads after enzyme digestion, purifying the magnetic beads to obtain a purified product II; performing a second round of PCR amplification on the purified product II to obtain a second round of PCR amplification products, adding a magnetic bead liquid to the second round of PCR amplification products, and obtaining the product after magnetic bead purification. The present invention amplifies the target region through multiple primers, quickly completes the construction of the sequencing library in a relatively short time, introduces a UMI label when constructing the sequencing library, and improves the detection sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene sequencing, and in particular relates to a method and a kit for constructing a multi-gene and multi-mutation type sequencing library for targeted treatment of lung cancer. Background Art

[0002] Lung cancer is currently the cancer with the highest morbidity and mortality in my country. Histopathological results can divide lung cancer into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which non-small cell lung cancer accounts for more than 85%. In recent years, with the rapid development of molecular technology and targeted drugs, precision therapy based on mutant genes has significantly improved the survival of NSCLC patients. Although traditional single-gene or single mutation type detection methods (such as PCR and fluorescence in situ hybridization) have certain sensitivity, their application is limited due to the complex molecular characteristics of lung cancer, involving multiple genes (such as EGFR, ALK, KRAS, TP53, etc.) and multiple mutation types (including point mutations, insertions / deletions, gene rearrangements, copy number variations, etc.). High-throughput sequencing technology (NGS) can comprehensively analyze multiple genes and mutation types in one test, greatly improving the detection efficiency and comprehensiveness of diagnosis. However, the current NGS technology mainly uses the probe hybridization capture method in the detection of multiple genes and multiple mutation types in lung cancer. The invention patent with application publication number CN107619867A discloses a sequence combination and probe for simultaneous detection of multiple gene mutation types in lung cancer. The capture process of the sequence combination and probe for simultaneous detection of multiple gene mutation types in lung cancer includes hybridization, washing and elution steps, and the library construction cycle is long, resulting in a long detection time. In addition, the probe capture method is not as sensitive as the multiplex amplification method for the detection of low-frequency mutations, and may be limited in low-input samples. Summary of the invention

[0003] The first objective of the present invention is to provide a method for constructing a multi-gene and multi-mutation type sequencing library for targeted treatment of lung cancer to solve the technical problems of long library construction cycle and long detection time in the prior art.

[0004] The second object of the present invention is to provide a kit for the targeted treatment of multiple genes and multiple mutations in lung cancer.

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

[0006] The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer comprises the following steps:

[0007] S1: A multiplex PCR amplification primer set is designed based on the targeted diagnostic gene, and the multiplex PCR amplification primer set is subjected to one round of PCR amplification to obtain one round of PCR amplification products; the multiplex PCR amplification primer set includes a DNA amplification primer and an RNA amplification primer; the DNA amplification primer includes a universal sequence and a DNA specific sequence, and the nucleotide sequence of the DNA specific sequence is shown in SEQ ID No.3-134; the RNA amplification primer includes a universal sequence and an RNA specific sequence, and the nucleotide sequence of the RNA specific sequence is shown in SEQ ID No.135-150; the universal sequence includes an F-terminal UMI universal sequence and an R-terminal universal sequence, and the nucleotide sequence of the F-terminal UMI universal sequence is shown in SEQ ID No.1, and the nucleotide sequence of the R-terminal universal sequence is shown in SEQ ID No.2;

[0008] S2: adding magnetic beads to the amplified product of one round of PCR in S1, purifying with magnetic beads to obtain purified product I with magnetic beads, followed by enzyme digestion;

[0009] S3: adding magnetic bead solution to the purified product I with magnetic beads after enzyme digestion in S2, and purifying with magnetic beads to obtain purified product II;

[0010] S4: Perform two rounds of PCR amplification on the purified product II in S3 to obtain a second round of PCR amplification product, add magnetic bead liquid to the second round of PCR amplification product, and obtain the product after magnetic bead purification.

[0011] Furthermore, the targeted diagnostic genes described in S1 are ALK, BRAF, EGFR, KRAS, MET, ERBB2, PIK3CA, NTRK1, NTRK3, ROS1, and RET.

[0012] Further, for the DNA-specific sequence designed for the KRAS gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.3-5, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.6-8; for the DNA-specific sequence designed for the ERBB2 gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.9-38, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.39-68; for the DNA-specific sequence designed for the PIK3CA gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.69-72, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.73-76; for the DNA-specific sequence designed for the EGFR gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.77-83, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.84-90; for the DNA-specific sequence designed for the MET gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.91-110, and the nucleotide sequence of the reverse primer of the DNA specific sequence is shown in SEQ ID No.111-130; the DNA specific sequence designed for the BRAF gene, the nucleotide sequence of the forward primer of the DNA specific sequence is shown in SEQ ID No.131-132, and the nucleotide sequence of the reverse primer of the DNA specific sequence is shown in SEQ ID No.133-134; the RNA specific sequence designed for the ALK gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.135-136, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.137-138; the RNA specific sequence designed for the ROS1 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.139, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.140; the RNA specific sequence designed for the RET gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.141, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.142; the RNA specific sequence designed for the NTRK3 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.143, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.144; RNA specific sequence designed for NTRK1 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.145-147, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.148-150. .

[0013] Furthermore, the multiplex PCR amplification primer set is optimized by excluding non-specific amplification primers.

[0014] Furthermore, the step of purifying the magnetic beads in S2, S3 and S4 is: washing with 80% by volume ethanol.

[0015] Furthermore, the volume of the PCR reaction system for one round of PCR amplification in S1 is 30 μL, including 10 μL of reverse transcription product, 1 μL of DNA amplification primer, 1.6 μL of RNA amplification primer, 10 μL of amplification buffer, and 7.4 μL of nuclease-free water; the method for preparing the reverse transcription product is: taking sample DNA, sample RNA, and 2 μL of reverse transcription reagent, and filling up to 10 μL with nuclease-free water to prepare a reverse transcription reaction system, and then performing a PCR reaction to obtain; the amplification program of the PCR reaction is: hot cover 105 ℃; 37 ℃, 15 min; 85 ℃, 5 s; 4 ℃ insulation; the concentration of the sample DNA is ≥5 ng / μL, and the concentration of the sample RNA is ≥5 ng / μL.

[0016] Furthermore, the amplification program of one round of PCR amplification described in S1 is: hot cover 105°C; 95°C, 3 min 30 s, one cycle; 98°C, 30 s, 62°C, 6 min, 72°C, 30 s, three cycles; 4°C, insulation.

[0017] Furthermore, the enzyme digestion reaction system includes 8 μL of purified product I with magnetic beads, 2 μL of digestion buffer, 1 μL of exonuclease I, and is supplemented to 20 μL with nuclease-free water; the reaction temperature of the enzyme digestion is 37° C., and the reaction time is 20 min.

[0018] Furthermore, the volume of the PCR amplification reaction system for the second-round PCR amplification in S4 is 30 μL, and the PCR amplification reaction system includes 8 μL of purified product II in S3, 1 μL of I5 Index, 1 μL of I7 Index, 10 μL of amplification buffer, and 10 μL of nuclease-free water; the amplification program for the second-round PCR amplification is: hot cover 105°C; 95°C, 3min30s, 1 cycle; 98°C, 20s, 80°C, 1s, 72°C, 45s, 76°C, 45s, 22 cycles; keep warm at 4°C; the temperature change rates of 80-72°C and 72-76°C in the amplification program of the second-round PCR amplification are both 0.1°C / s.

[0019] A kit for targeted treatment of lung cancer with multiple genes and multiple mutations includes the above-mentioned multiplex PCR amplification primer set.

[0020] Beneficial effects of the present invention:

[0021] The present invention selects targets based on clinical data, and the target selection is most relevant to the target drug, so that the target drug can cover 404 relevant mutations of ALK, BRAF, EGFR, KRAS, MET, ERBB2, PIK3CA, NTRK1, NTRK3, ROS1, and RET genes.

[0022] The present invention uses PCR amplification to enrich the target fragments, and the sequencing depth uniformity of the amplicons is above 95%. When the multi-gene multi-mutation type sequencing library for targeted lung cancer treatment of the present invention is established, it is only necessary to extract all the target fragments of the sample once, and no additional RNA sample needs to be extracted for the detection of gene fusion, thereby reducing sample waste.

[0023] The method for constructing a multi-gene and multi-mutation type sequencing library for targeted treatment of lung cancer of the present invention starts from one tube of sample nucleic acid, and DNA and RNA are operated in the same tube throughout the whole process, which reduces the loss of target fragments, avoids the possibility of cross contamination, and reduces the complexity of the experiment.

[0024] The present invention uses multiple primers to amplify the target region and quickly completes the construction of a multi-gene and multi-mutation type sequencing library for targeted treatment of lung cancer in a relatively short period of time. The introduction of UMI tags when constructing the sequencing library can not only more effectively detect low-abundance mutations and improve detection sensitivity, but also better remove amplification bias, thereby improving detection accuracy.

[0025] The present invention solves the problems of long time consumption, low detection sensitivity and high cost in the prior art of sequencing library establishment, and improves the timeliness, sensitivity and accuracy of gene detection. The results of clinical sample performance verification show that the minimum detection limit of single base variation / insertion and deletion in tissue samples reaches 0.5%, gene rearrangement reaches 50 copies, copy number variation reaches 3 copies, and the minimum detection limit of mutation in blood cfDNA samples reaches 0.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart for detection of different mutation types;

[0027] Figure 2 It is an optimization diagram of the one-step reverse transcription process in Example 1, wherein A is the experimental process time of the one-step reverse transcription process and the two-step reverse transcription process, B is a sequencing library concentration diagram of the one-step reverse transcription process and the two-step reverse transcription process, and C is a fragment distribution diagram of the sequencing library of the one-step reverse transcription process and the two-step reverse transcription process;

[0028] Figure 3 Optimize the exclusion map for non-specific amplification primers (in charge of primer library fragment distribution);

[0029] Figure 4 Primer optimization exclusion plot for nonspecific amplification (fragment distribution of a single primer pair library). DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0031] Example 1

[0032] The method for constructing a multi-gene multi-mutation type sequencing library for lung cancer in Example 1 comprises the following steps:

[0033] 1. Sample extraction

[0034] Paraffin tissue samples were used as samples. The RNeasy FFPE Kit (QIAGEN, #73504) was used to extract sample DNA and sample RNA according to the instructions, and the concentration was measured using a Qubit4 fluorometer.

[0035] 2. Reverse transcription reaction

[0036] Prepare the reverse transcription reaction system in a 0.2 mL enzyme-free PCR tube according to Table 1, use a pipette to blow or shake, centrifuge the reverse transcription reaction system to the bottom of the enzyme-free PCR tube, and perform PCR reaction according to the PCR amplification program in Table 1 to obtain the reverse transcription product. The sample DNA concentration in the reverse transcription reaction system is ≥5 ng / μL, and the sample RNA concentration is greater than the sample DNA concentration. Figure 1 The reverse transcription reaction in the embodiment 1 is a one-step reverse transcription reaction. Figure 2 It can be seen that compared with the conventional two-step reverse transcription, the one-step reverse transcription takes less time and the resulting sequencing library has a higher concentration.

[0037] Table 1 Reverse transcription reaction system and amplification procedure

[0038]

[0039] 3. One round of PCR amplification (targeted amplification reaction)

[0040] Add the one-round PCR amplification reaction system in Table 2 to the 0.2 μL enzyme-free PCR tube in step 2, then use a pipette to blow or oscillate to mix, centrifuge the one-round PCR amplification reaction system to the bottom of the enzyme-free PCR tube, and perform one-round PCR amplification according to the amplification program of the one-round PCR amplification reaction system in Table 2 to obtain one-round PCR amplification products.

[0041] DNA amplification primers were optimized by eliminating non-specific amplification primers. The specific method is as follows: DNA primers (DNA Primer) were divided into 8 tubes, each tube containing 8-10 pairs of primers, and the 8 tubes of DNA primers were mixed with RNA primers (RNA Primer) for library construction experiments. Figure 3 As shown, Figure 3 The red box marked position, that is, DNA Primer 43-50, showed non-specific amplification. Then, DNA Primer 43-50 was divided into 8 tubes, and each tube was mixed with RNA Primer to build a library. Figure 4 As shown, Figure 4 The position marked by the red box, i.e., DNA Primer 50, has non-specific primer amplification. DNA Primer 50 is the amplicon primer of MET exon 13, which is used to detect the copy number variation of the MET gene. Because the primers of other exons of MET exist, removing DNA Primer 50 will not affect the CNV detection of MET. The nucleotide sequence of DNA Primer 50 is shown in SEQ ID No.151-152. The specific components of the amplification buffer are: Tris Buffer (PH 8.5, 40 mM), MgCl2 (5 mM), dNTP (0.2 mM), Taq polymerase (0.1 U / μL), H2O.

[0042] Table 2 One-round PCR amplification reaction system and amplification procedure

[0043]

[0044] 4. First magnetic bead purification

[0045] 4.1. Add 30 μL of magnetic bead solution to the enzyme-free PCR tube containing the product of a round of PCR amplification obtained in step 3, and mix by gently pipetting 20 times;

[0046] 4.2. Incubate at room temperature for 10 min, and place the enzyme-free PCR tube on the DynaMag -96 Side magnetic stand for 3 min.

[0047] 4.3. Remove the supernatant and add 180 μL of 80% ethanol solution to the enzyme-free PCR tube and let stand for 30 seconds.

[0048] 4.4. Repeat step 4.3 once;

[0049] 4.5. Let stand at room temperature for 3 min to allow the ethanol to evaporate;

[0050] 4.6. Add 24 μL of nuclease-free water to the enzyme-free PCR tube, gently use a pipette to resuspend the magnetic beads to avoid bubbles, and let stand at room temperature for 2 min to obtain the purified product I with magnetic beads, and perform enzyme digestion on the purified product I with magnetic beads;

[0051] 5. Enzymatic digestion of purified product I with magnetic beads.

[0052] The enzyme digestion reaction system in Table 3 was added to a 0.2 mL enzyme-free PCR tube and reacted at 37 °C for 20 min to obtain the purified product I with magnetic beads after enzyme digestion. The digestion buffer included 670 mM glycine-KOH (pH 9.5, 25 °C), 67 mM MgCl2, and 10 mM dithiothreitol (DTT).

[0053] Table 3 Enzyme digestion reaction system

[0054] Reagents Volume (μL) Purification product with magnetic beads Ⅰ 8 Digestion buffer 2 Exonuclease I 1 Nuclease-free water 9

[0055] 6. Second round of magnetic bead purification

[0056] 6.1. Add 25 μL of magnetic bead solution to the purified product I with magnetic beads obtained after enzyme digestion in step 5, and mix gently by pipetting 20 times;

[0057] 6.2. Incubate at room temperature for 10 min, and place the enzyme-free PCR tube on the DynaMag -96 Side magnetic stand for 3 min;

[0058] 6.3. Remove the supernatant and add 180 μL of 80% ethanol solution to the enzyme-free PCR tube and let stand for 30 seconds.

[0059] 6.4. Repeat step 6.3 once;

[0060] 6.5. Let stand at room temperature for 3 min to allow the ethanol to evaporate;

[0061] 6.6. Remove the centrifuge tube from the magnetic rack, add 10 μL of nuclease-free water, mix gently by pipetting 20 times, resuspend the magnetic beads to avoid bubbles, and let stand at room temperature for 2 min;

[0062] 6.7. Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0063] 6.8. Use a pipette to aspirate 8 μL of supernatant and transfer it to a new PCR tube to obtain 8 μL of purified product II.

[0064] 7. Second round of PCR amplification

[0065] Add the second-round PCR amplification reaction system in Table 4 to the enzyme-free PCR tube, then gently blow or shake with a pipette to mix, centrifuge the amplification reaction system to the bottom of the tube, and perform second-round PCR amplification according to the amplification program in Table 4 to obtain the second-round PCR amplification product. The specific components of the amplification buffer are: Tris Buffer (PH 8.5, 40mM), MgCl2 (5mM), dNTP (0.2mM), Taq polymerase (0.1U / μL), H2O.

[0066] Table 4 Second round PCR amplification reaction system and amplification procedure

[0067]

[0068] Ramping*: The temperature ramp from 80℃ to 72℃ and from 72℃ to 76℃ should be set to 0.1℃ / s.

[0069] 8. The third round of magnetic bead purification

[0070] 8.1. Add 30 μL of purified magnetic beads to the second-round PCR amplification product obtained in step 7, and mix gently by pipetting 20 times;

[0071] 8.2. Incubate at room temperature for 10 min, and place the enzyme-free PCR tube on the DynaMag -96 Side magnetic stand for 3 min.

[0072] 8.3. Remove the supernatant and add 180 μL of 80% ethanol solution to the enzyme-free PCR tube and let stand for 30 seconds.

[0073] 8.4. Repeat step 8.3 once;

[0074] 8.5. Let stand at room temperature for 3 min to allow the ethanol to evaporate;

[0075] 8.6. Add 24 μL of nuclease-free water to the enzyme-free PCR tube, gently pipette to resuspend the magnetic beads, avoid bubbles, and let stand at room temperature for 2 min;

[0076] 8.7. Place the PCR tube back on the magnetic rack and let it stand for 3 minutes;

[0077] 8.8. Use a pipette to aspirate 20 μL of supernatant and transfer it to a new PCR tube to obtain a multi-gene and multi-mutation sequencing library for targeted treatment of lung cancer.

[0078] 9. Library quality inspection

[0079] The multi-gene multi-mutation sequencing library for targeted therapy of lung cancer was measured using Qubit® 4.0 (Qubit dsDNA HSAssay Kit) and Qsep100 for library concentration and fragment length. The concentration range of a normal library is 1 ng / μL-15ng / μL, and the main peak fragment distribution range is 240-270 bp.

[0080] 10. Result Analysis

[0081] 10.1. SNV (single base variation) / Indel (insertion and deletion) site detection

[0082] Table 5 SNV / Indel site detection

[0083]

[0084] 10.2. Fusion (gene rearrangement) fusion site detection

[0085] Table 6 Fusion site detection

[0086]

[0087] 10.3. CNV (copy number variation) site detection

[0088] Table 7 CNV site detection

[0089]

[0090] 10.4. Detection of SNV / Indel sites in ctDNA samples

[0091] Table 8 SNV / Indel site detection in ctDNA samples

[0092]

[0093] Table 9 DNA amplification primers

[0094] Gene F-terminal specific primer R-specific primer Remark DNAPrimer 1 KRAS SEQ ID No.3 TAGCTGTATCGTCAAGGCACTC SEQ ID No.6 AAGGCCTGCTGAAAATGACTGA SNV / Indel Primers DNAPrimer 2 ERBB2 SEQ ID No.9 ATCCTGCCCTTTGCCCAAC SEQ ID No.39 ACACTGCTCATGGCAGCAG CNV related primers DNAPrimer 3 ERBB2 SEQ ID No.10 GGTAAGGTGCCCACCTTTCTC SEQ ID No.40 CAGTTCCCTCAGTGAGCGC CNV related primers DNAPrimer 4 ERBB2 SEQ ID No.11 TCATCCACCATAACACCCACCT SEQ ID No.41 AGTGTGGAGCAGAGCTTGGT CNV related primers DNAPrimer 5 ERBB2 SEQ ID No.12 TCTCCTACATGCCCATCTGGAA SEQ ID No.42 TGCAGAAAAGACCGTTGGACTC CNV related primers DNAPrimer 6 ERBB2 SEQ ID No.13 TGTGTGGACCTGGATGACAAG SEQ ID No.43 AGTGAAGGGCAATGAAGGGTAC CNV related primers DNAPrimer 7 ERBB2 SEQ ID No.14 TGTGAAAATTCCAGTGGCCATC SEQ ID No.44 CTAGCAGGAGAGGGTGGAGG CNV related primers DNAPrimer 8 ERBB2 SEQ ID No.15 CAGTTGGAGGACTTCCTCTTCTG SEQ ID No.45 ACACATCACTCTGGTGGGTGA CNV related primers DNAPrimer 9 ERBB2 SEQ ID No.16 CTGTCTCTGCCTTAGGTGTGAC SEQ ID No.46 TTCCAGCAGGTCAGGGATCT CNV related primers DNAPrimer10 ERBB2 SEQ ID No.17 TCCTTCCTCCACAGAATGAGGA SEQ ID No.47 TGTCATCGTCCTCCAGCAGT CNV related primers DNAPrimer11 ERBB2 SEQ ID No.18 AGCCTTCGACAACCTCTATTACTG SEQ IDNo.48 TAGGTGTCCCTTTGAAGGTGCT CNV scientists DNAPrimer12 PIK3CA SEQ IDNo.69 GAATGTTTACTACCAAATGGAATGATAGTGA SEQ IDNo.73 CTTGCTTCTTTAAATAGTTCATGCTTTATGG SNV / Indel Copyright DNAPrimer13 PIK3CA SEQ IDNo.70 CAGCTCAAAGCAATTTCTACACGA SEQ IDNo.74 CCATTTTAGCACTTACCTGTGACTC SNV / Indel Copyright DNAPrimer14 PIK3CA SEQ IDNo.71 GCAGCCATTGACCTGTTTACAC SEQ IDNo.75 TCACCATGATGTTACTATTGTGACG SNV / Indel Copyright DNAPrimer15 EGFR SEQ IDNo.77 CCACACTGACGTGCCTCTC SEQ IDNo.84 AGGTGAGGCAGATGCCCA SNV / Indel Copyright DNAPrimer16 EGFR SEQ IDNo.78 GCCAGTTAACGTCTTCCTTCTCT SEQ IDNo.85 CACACAGCAAAGCAGAAACTCA SNV / Indel Copyright DNAPrimer17 PIK3CA SEQ IDNo.72 ACTACAATCTTTTGATGACATTGCA SEQ IDNo.76 ATCCATTTTTGTTGTCCAGCCA SNV / Indel Copyright DNAPrimer18 ERBB2 SEQ IDNo.19 TCTCAGCGTACCCTTGTCCC SEQ IDNo.49 GCAAAGAGCCCAGGTGCATA CNV license plate,SNV / Indel license plate DNAPrimer19 ERBB2 SEQ IDNo.20 AACTACCTTTCTACGGACGTGG SEQ IDNo.50 CTCGGGCACAGGGCTTG CNV license plate,SNV / Indel license plate DNAPrimer20 ERBB2 SEQ IDNo.21 TAGCGGAGCGATGCCCAA SEQ IDNo.51 ACCTGGCCCTGACCTTGTAG CNV scientists DNAPrimer21 ERBB2 SEQ IDNo.22 CTGCCTCCACTTCAACCACA SEQ IDNo.52 GACAGGCAGTCACACAGCT CNV scientists DNAPrimer22 ERBB2 SEQ ID No. 23 GGGATGAGCTACCTGGAGGA SEQ ID No. 53 AACCTTGCCCCCATCTGC CNV - related primer DNA Primer 23 ERBB2 SEQ ID No. 24 TGGCTTCCGCCCCCA SEQ ID No. 54 TCACCCCGCCTCACCTC CNV - related primer DNA Primer 24 ERBB2 SEQ ID No. 25 CCACGGGGCCCTTTACTG SEQ ID No. 55 CATGGCTCCGGCTGGAC CNV - related primer DNA Primer 25 ERBB2 SEQ ID No. 26 CCGGCACAGACATGAAGCT SEQ ID No. 56 AGGTTTCCCTGCACCACCT CNV - related primer DNA Primer 26 ERBB2 SEQ ID No. 27 CACCCAGCTCTTTGAGGACA SEQ ID No. 57 CTGTGACAGGGGTGGTATTGT CNV - related primer DNA Primer 27 ERBB2 SEQ ID No. 28 TGAAAGGAGGGGTCTTGATCCA SEQ ID No. 58 GAGAGCCAGCTGGTTGTTCTT CNV - related primer DNA Primer 28 ERBB2 SEQ ID No. 29 AGACATCTCTCTCACTGCCTGT SEQ ID No. 59 ATCCTCAGAACTCTCTCCCCAG CNV - related primer DNA Primer 29 ERBB2 SEQ ID No. 30 ACCTCCTCAGTGTGCTATGGT SEQ ID No. 60 GATCTTCTTGCAGCCAGCAAAC CNV - related primer DNA Primer 30 ERBB2 SEQ ID No. 31 GACCCAGCCTCCAACACTG SEQ ID No. 61 GATGCAGAGACAGAGCCCAC CNV - related primer DNA Primer 31 ERBB2 SEQ ID No. 32 GCCTGCAGGTTACCTATACATCTC SEQ ID No. 62 CAGAATTCGTCCCCGGATTACTT CNV - related primer DNA Primer 32 ERBB2 SEQ ID No. 33 TGGCCTGCCACCAGCT SEQ ID No. 63 GAAGGAACTGGCTGCAGTTGA CNV - related primer DNA Primer 33 ERBB2 SEQ ID No. 34 CCCCAGGGAGTATGTGAATGC SEQ ID No. 64 TCCAAAACAGGTCACTGAGCC CNV-related primers DNAPrimer34 ERBB2 SEQ ID No.35 TCTGAATGTCGGCCAAGATTCC SEQ ID No.65 TACCTGGATGACCACAAAGCG CNV-related primers DNAPrimer35 ERBB2 SEQ ID No.36 GGACCTGGTGGATGCTGAG SEQ ID No.66 ACCATGCCCCCAGCG CNV-related primers DNAPrimer36 ERBB2 SEQ ID No.37 TTTGATGGTGACCTGGGAATGG SEQ ID No.67 TCCTCACTGTACCGCTGTAGA CNV-related primers DNAPrimer37 ERBB2 SEQ ID No.38 TGACTTCTGCTGGCATCAAGAG SEQ ID No.68 CAAGCAGGAAGGAAGGTTCCTT CNV-related primers DNAPrimer38 MET SEQ ID No.91 AGGGCGCGAGCCAGA SEQ ID No.111 GAGAAGTCAGCGGCGCAA CNV-related primers DNAPrimer39 MET SEQ ID No.92 CCGGTTCATCAACTTCTTTGTAGG SEQ ID No.112 CGTTTCCTTTAGCCTTCTCACTG CNV-related primers DNAPrimer40 MET SEQ ID No.93 CAGCGCGTTGACTTATTCATGG SEQ ID No.113 CCAAGATTAGCTATGGTGAGGTCTC CNV-related primers DNAPrimer41 MET SEQ ID No.94 CCCTCATGTGAATTTTCTCCTGGA SEQ ID No.114 CAGTGATAACCAGTGTGTAGCCAT CNV-related primers DNAPrimer42 MET SEQ ID No.95 GACATTTCCAGTCCTGCAGTCA SEQ ID No.115 GATCGCACACATTTGTCGTGG CNV-related primers DNAPrimer43 MET SEQ ID No.96 CATATGTGGCTGGGACTTTGGA SEQ ID No.116 TGCAGCTCTCATTTCCAAGGAG CNV-related primers DNAPrimer44 MET SEQ ID No.97 CCAGATTGAAATGCACAGTTGGT SEQ ID No.117 TGTCCCGTGGCCATTTGAAATA CNV-related primers DNAPrimer45 MET SEQ ID No.98 ATTTCGCCGAAATACGGTCCTA SEQ ID No.118 TGTGTCTAGAATTCCCACTGTTTAGG CNV - related primer DNA Primer46 MET SEQ ID No.99 GTTATACCCCAGCCCAAACCAT SEQ ID No.119 CTGAAGATGCTTGTCTCTCGGT CNV - related primer DNA Primer47 MET SEQ ID No.100 AGTGGTGGGAGCACAATAACAG SEQ ID No.120 GTTCCTTCCTGCTTCATGCACA CNV - related primer DNA Primer48 MET SEQ ID No.101 GAAAACCAAAGCCTTTTTCATGTTAGATG SEQ ID No.121 ACTGGCTTTTCAAAAGGCTTAAACA CNV - related primer DNA Primer49 MET SEQ ID No.102 GACCCTGAAGCAGTTAAAGGTGA SEQ ID No.122 TTGGGGACCGTGCATAAAACG CNV - related primer DNA Primer50 MET SEQ ID No.151 TCAACCAGATCAGAATTTCACAGGA SEQ ID No.152 TCTCTTTTTCAGCCACAGGAAAAAC CNV - related primer, resulting in non - specific amplification DNA Primer51 MET SEQ ID No.103 CATCCTAACTAGTGGGGACTCTGA SEQ ID No.123 CTCTGGATTTAGAGCACTGAGGTC CNV - related primer DNA Primer52 MET SEQ ID No.104 GTTCTTTCTTTTGCACAGGGCAT SEQ ID No.124 ACAGCACAGTGAATTTTCTTGCC CNV - related primer DNA Primer53 MET SEQ ID No.105 TCTGACCGAGGGAATCATCATG SEQ ID No.125 CCTTCACTTCGCAGGCAGA CNV - related primer DNA Primer54 MET SEQ ID No.106 CTTTGGTCTTCAAGTAGCCAAAGG SEQ ID No.126 CAGTTTCTTGCAGCCAAGTCTC CNV - related primer DNA Primer55 MET SEQ ID No.107 GCTGATTTTGGTCTTGCCAGAG SEQ ID No.127 TCCAAAGCCATCCACTTCACTG CNV - related primer DNA Primer56 MET SEQ ID No. 108 CCCACCTTATCCTGACGTAAACAC SEQ ID No. 128 TCTGGGCAGTATTCGGGTTGTA CNV - related primers DNA Primer 57 MET SEQ ID No. 109 AGTTGTCATATAAAAACCCACTGTTTGAG SEQ ID No. 129 ACACACAAAACATCTACATGTTCACATT CNV - related primers DNA Primer 58 MET SEQ ID No. 110 TCGTCGATTCTTGTGTGCTGTC SEQ ID No. 130 GTAGCGAACTAATTCACTGCCCA CNV - related primers, SNV / Indel primers DNA Primer 59 KRAS SEQ ID No. 4 TGCAGAAAACAGATCTGTATTTATTTCAGT SEQ ID No. 7 ACAGGCTCAGGACTTAGCAAGA SNV / Indel primers DNA Primer 60 KRAS SEQ ID No. 5 GTACTGGTCCCTCATTGCACTG SEQ ID No. 8 CTTCTCAGGATTCCTACAGGAAGC SNV / Indel primers DNA Primer 61 EGFR SEQ ID No. 79 TGGCTGGTTATGTCCTCATTGC SEQ ID No. 86 GACTGCTAAGGCATAGGAATTTTCG SNV / Indel primers DNA Primer 62 EGFR SEQ ID No. 80 CATAGGTCTGCCGCAAATTCC SEQ ID No. 87 ACTCACGGGGACACTTCTTCA SNV / Indel primers DNA Primer 63 BRAF SEQ ID No. 131 ACCATGCCACTTTCCCTTGTAG SEQ ID No. 133 AGACGGGACTCGAGTGATGATT SNV / Indel primers DNA Primer 64 EGFR SEQ ID No. 81 CTCCCAACCAAGCTCTCTTGAG SEQ ID No. 88 TGTGCCAGGGACCTTACCTTAT SNV / Indel primers DNA Primer 65 EGFR SEQ ID No. 82 CCTCACCTCCACCGTGCA SEQ ID No. 89 TTGCGATCTGCACACACCA SNV / Indel primers DNA Primer 66 EGFR SEQ ID No. 83 GTTTCAGGGCATGAACTACTTGG SEQ ID No. 90 AAGCCACCTCCTTACTTTGCC SNV / Indel Primer DNAPrimer67 BRAF SEQ ID No.132 TGATGGGACCCACTCCATCG SEQ ID No.134 GATCTACTGTTTTCCTTTACTTACTACACC SNV / Indel Primer

[0095] Table 10 RNA amplification primers

[0096] F - end Specific Primer R - end Specific Primer Fusion Site - corresponding Primer SEQ ID No.135 AACTACTGTAGAGCCCACACCT SEQ ID No.137 AGCTTGCTCAGCTTGTACTCAG EML4--ALK_chr2:42295516:+_chr2:29223528:-(1) SEQ ID No.136 TCTCAACCAGCAAAAATGTCAACTC SEQ ID No.138 TCTGCAGCTCCATCTGCATG EML4--ALK_chr2:42325554:+_chr2:29223528:-(3), Clinical sample BLK - 8, Clinical sample BLK - 12 SEQ ID No.139 TTGGAGCAAAAGCCCACTGA SEQ ID No.140 AGGTCAGTGGGATTGTAACAACC CD74--ROS1_chr5:150404680:-_chr6:117324415:-(5), Clinical sample BLK - 13 SEQ ID No.141 CCTTGCAGAAATAGGAATTGCTGTG SEQ ID No.142 GTTGCCTTGACCACTTTTCCAAA KIF5B--RET_chr10:32028428:-_chr10:43116584:+(8), Clinical sample BLK - 14 SEQ ID No.143 ACGCGTGATCCAGCTGATG SEQ ID No.144 AAGGCTCCCTCACCCAGTT ETV6--NTRK3_chr12:11869969:+_chr15:87940753:-(11) SEQ ID No.145 TTGGGTTTTTCCTGTGGCTGA SEQ ID No.148 ACTGCACTTGTCGGCATGAA MET SEQ ID No.146 GGTTTTTCCTGTGGCTGAAAAAGAG SEQ ID No.149 GTCTGTCAGAGGATACTGCACTTG MET SEQ ID No.147 GTTTTTCCTGTGGCTGAAAAAGAGA SEQ ID No.150 GACATGTCTGTCAGAGGATACTGC MET

[0097] Table 11 Universal sequences

[0098] Sequence F - end UMI Universal Sequence SEQ ID No.1 GGACACTCTTTCCCTACACGACGCTCTTCCGATCTnnnnnnnnnnATGGGAAAGAGTGTCC Common sequence at the R end SEQ ID No.2 GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA

Claims

1. A method for constructing a multi-gene and multi-mutation type sequencing library for targeted treatment of lung cancer, characterized in that: The following steps are involved: S1: A multiplex PCR amplification primer set is designed based on the targeted diagnostic gene, and the multiplex PCR amplification primer set is subjected to one round of PCR amplification to obtain one round of PCR amplification products; the multiplex PCR amplification primer set includes a DNA amplification primer and an RNA amplification primer; the DNA amplification primer includes a universal sequence and a DNA specific sequence, and the nucleotide sequence of the DNA specific sequence is shown in SEQ ID No.3-134; the RNA amplification primer includes a universal sequence and an RNA specific sequence, and the nucleotide sequence of the RNA specific sequence is shown in SEQ ID No.135-150; the universal sequence includes an F-terminal UMI universal sequence and an R-terminal universal sequence, and the nucleotide sequence of the F-terminal UMI universal sequence is shown in SEQ ID No.1, and the nucleotide sequence of the R-terminal universal sequence is shown in SEQ ID No.2; S2: adding magnetic beads to the amplified product of one round of PCR in S1, purifying with magnetic beads to obtain purified product I with magnetic beads, followed by enzyme digestion; S3: adding magnetic bead solution to the purified product I with magnetic beads after enzyme digestion in S2, and purifying with magnetic beads to obtain purified product II; S4: Perform two rounds of PCR amplification on the purified product II in S3 to obtain a second round of PCR amplification product, add magnetic bead liquid to the second round of PCR amplification product, and obtain the product after magnetic bead purification.

2. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The targeted diagnostic genes described in S1 are ALK, BRAF, EGFR, KRAS, MET, ERBB2, PIK3CA, NTRK1, NTRK3, ROS1, and RET.

3. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 2, characterized in that: The DNA-specific sequence designed for the KRAS gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.3-5, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.6-8; the DNA-specific sequence designed for the ERBB2 gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.9-38, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.39-68; the DNA-specific sequence designed for the PIK3CA gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.69-72, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.73-76; the DNA-specific sequence designed for the EGFR gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.77-83, and the nucleotide sequence of the reverse primer of the DNA-specific sequence is shown in SEQ ID No.84-90; the DNA-specific sequence designed for the MET gene, the nucleotide sequence of the forward primer of the DNA-specific sequence is shown in SEQ ID No.91-110, the nucleotide sequence of the reverse primer of the DNA specific sequence is shown in SEQ ID No.111-130; the DNA specific sequence designed for the BRAF gene, the nucleotide sequence of the forward primer of the DNA specific sequence is shown in SEQ ID No.131-132, and the nucleotide sequence of the reverse primer of the DNA specific sequence is shown in SEQ ID No.133-134; the RNA specific sequence designed for the ALK gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.135-136, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.137-138; the RNA specific sequence designed for the ROS1 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.139, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.140; the RNA specific sequence designed for the RET gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.141, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No. ID No.142; the RNA specific sequence designed for the NTRK3 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.143, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.144; RNA specific sequence designed for NTRK1 gene, the nucleotide sequence of the forward primer of the RNA specific sequence is shown in SEQ ID No.145-147, and the nucleotide sequence of the reverse primer of the RNA specific sequence is shown in SEQ ID No.148-150. .

4. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The multiplex PCR amplification primer set is optimized by excluding non-specific amplification primers.

5. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The steps of magnetic bead purification described in S2, S3 and S4 are: washing with 80% by volume ethanol.

6. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The volume of the PCR reaction system for one round of PCR amplification described in S1 is 30 μL, including 10 μL of reverse transcription product, 1 μL of DNA amplification primer, 1.6 μL of RNA amplification primer, 10 μL of amplification buffer, and 7.4 μL of nuclease-free water; the method for preparing the reverse transcription product is: taking sample DNA, sample RNA, and 2 μL of reverse transcription reagent, and filling up to 10 μL with nuclease-free water to prepare the reverse transcription reaction system, and then performing a PCR reaction to obtain; the amplification program of the PCR reaction is: hot cover 105 ℃; 37 ℃, 15 min; 85 ℃, 5 s; 4 ℃ insulation; the concentration of the sample DNA is ≥5 ng / μL, and the concentration of the sample RNA is ≥5 ng / μL.

7. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The amplification program for one round of PCR amplification described in S1 is: heated cover 105°C; 95°C, 3 min 30 s, one cycle; 98°C, 30 s, 62°C, 6 min, 72°C, 30 s, three cycles; 4°C, insulation.

8. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The enzyme digestion reaction system includes 8 μL of purified product I with magnetic beads, 2 μL of digestion buffer, 1 μL of exonuclease I, and is supplemented to 20 μL with nuclease-free water; the reaction temperature of the enzyme digestion is 37° C., and the reaction time is 20 min.

9. The method for constructing a multi-gene multi-mutation type sequencing library for targeted treatment of lung cancer according to claim 1, characterized in that: The volume of the PCR amplification reaction system for the second-round PCR amplification in S4 is 30 μL, and the PCR amplification reaction system includes 8 μL of purified product II in S3, 1 μL of I5 Index, 1 μL of I7 Index, 10 μL of amplification buffer, and 10 μL of nuclease-free water; the amplification program for the second-round PCR amplification is: hot cover 105°C; 95°C, 3 min 30 s, 1 cycle; 98°C, 20 s, 80°C, 1 s, 72°C, 45 s, 76°C, 45 s, 22 cycles; keep warm at 4°C; the temperature change rates of 80-72°C and 72-76°C in the amplification program of the second-round PCR amplification are both 0.1°C / s.

10. A kit for targeted treatment of lung cancer with multiple genes and multiple mutations, characterized in that: Comprising the multiplex PCR amplification primer set according to claim 1.

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