CN112029865A - Probe group and gene chip for simultaneously detecting multiple gene mutation types and application thereof - Google Patents
Probe group and gene chip for simultaneously detecting multiple gene mutation types and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene detection, and particularly relates to a probe set for simultaneously detecting multiple gene mutation types. The invention can detect mutation of up to 102 genes at one time, thereby finding known and/or unknown mutation; the invention can detect related rare hotspot variation, namely 14 exon skipping mutation of MET gene, BCL2L11(BIM)2 intron deletion polymorphism related region and NTRK1/2/3 hotspot fusion mutation at one time; the invention can detect known hot spot fusion variation and unknown fusion variation of 6 genes-ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 of specific fusion breakpoint regions more sensitively (0.5%).
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a probe set for simultaneously detecting multiple gene mutation types and application thereof.
Background
According to the World Health Organization (WHO) statistics, about 60% of people die from cancer, diabetes, cardiovascular disease, chronic respiratory disease, 4 major causes of death in the world. In 2015, about 392.9 ten thousand people suffer from Chinese malignant tumor, and about 233.8 ten thousand people die. On average, over 1 million people per day were diagnosed with cancer, and 7.5 people per minute were diagnosed with cancer. In recent decades, the onset and death of malignant tumors have been rising, and the medical cost of malignant tumors is over 2200 billion per year (Chen, W., et al., Cancer standards in China, 2015.CA Cancer J Clin, 2016.66 (2): p.115-32).
The development and development of tumors is a complex process involving multiple factors, multiple steps, and multiple genes. The research of tumor genetics reveals nearly 140 driving genes, and the common genes are: EGFR, KRAS, EML4-ALK, BRAF, PI3KCA, AKT1, MEK1, MET, HER2, RET, NRAS, and NTRK1, etc., which are distributed in 12 signaling pathways and regulate cell proliferation, apoptosis, and cell differentiation, respectively (Vogelstein, B., et al, Cancer gene landscapes. science, 2013.339 (6127): p.1546-58).
In the beginning of 2015, the global system of the united states, oarbama, officially started an 'accurate medical plan' in the statement of national consultancy, the current tumor treatment is gradually changed from the treatment of 'symptoms' to the treatment of 'genes', and appropriate targeted drugs are pertinently selected through mutation detection of related driving genes, so that individualized medical treatment is realized, and the optimal treatment effect of patients is achieved. By 2016, 72 targeted antineoplastic drugs were approved by the U.S. Food and Drug Administration (FDA). However, detection of tumor-driving genes is not straightforward. On one hand, the types of molecular targets involved are complex, including point mutation, short fragment insertion deletion, copy number variation, fusion gene and the like, and the omission of mutation types of related targets greatly reduces the targeted medication guidance of patients. On the other hand, tissue samples of patients with advanced tumors are difficult to obtain, and ctDNA (Cell-free Circulating Tumor DNA) detection is noninvasive, but due to the presence of a large amount of normal tissue free DNA, the ctDNA content is usually between 1% and 0.01% of ultralow frequency, and the precision detection is difficult. Currently, the most common technical method for detecting the driver gene is RT-PCR technology, and although the method is simple and rapid, the method has the limitations that only known mutation sites can be detected, the detected mutation types are not complete, the sensitivity is low (about 1 percent), and the like. The currently accepted gene mutation detection standard in the industry is a Sanger sequencing method, and the technology has the advantages of complex operation, low flux, low sensitivity (10%), high detection leakage risk and difficulty in meeting the detection requirements of a large number of tumor patients.
Therefore, there is a need in the art for a new technology that is comprehensive, highly sensitive, high-throughput, and simultaneously and precisely detects multiple mutation types, so as to effectively complement and improve the existing tumor personalized diagnosis and treatment.
Disclosure of Invention
As described above, there is a need in the art for a new technique that is comprehensive, highly sensitive, high-throughput, and simultaneously accurate for the detection of multiple mutation types.
Accordingly, in a first aspect, the present invention provides a probe set for simultaneously detecting multiple gene mutation types, the probe set comprising a probe for a targeted medication-related gene, wherein the targeted medication-related gene comprises EGFR, BRAF, KRAS, HER2, MET, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3, and gene mutations thereon comprise:
1) for the EGFR gene: g719, A698, V765, F712, L703, A702, G724, E709, S720, E709, S752_ I759del, L747_ S752del, L747_ T751del, E746_ A750del, E746_ S752> 747_ E749del, L747_ T751> 746_ E749del, K745_ E749del, L747_ T751del, S752_ I759del, T751_ I759del, L747_ P753> 746_ S752del, E _ T751del, E746_ T746 > 871 _ T871, G779D 793, G793D 793, G79779, G793D 770, G793D 770, 3D 793D 79, G793D 79, D793, D793D 79, D769, 3D 770, 3D 793D 79, 3D 793, 3D 79, K752 > 751, 3D 793D 7944, 3D 7944, 3D 770, 3D 7944, 3D 7944, 3D 78, 3D 7944, 3D 7944, 3D 793, 3D 79;
2) for the BRAF gene: a598_ T599insV, a598V, D594G, D594N, D594V, E586E, E586K, F583F, F595L, F595S, G596D, G596R, G606E, H608R, I582M, I592M, I592V, K601V, L584V, L597V, N593672, P213V, S605V, S V, T599_ V600insT, T599_ V600insTT, T599V, V600_ K601 593672 > V, V600, V36600, V600V V, V36600V V, V;
3) for the KRAS gene: g _ a11insG, G13insG, L19, G13, V14, G12, L19, G _ V14insG, G12, V8, a11, G12, G13, G15, a18, Q22, G13, a _ G12insG, a59, Q61, T58, a146, K117;
4) for the HER2 gene: D769H, L755S, I767M, L755P, G776> VC, V777L, G776V, G778_ S779insG, V777A, V842I, H878Y;
5) for the MET gene: exon skipping mutations of E168D, T1010I, H1112Y, H1124D, H1112L, L1130L, N1118Y, H1112R, M1268T, Y1248C, Y1253D, 14;
6) for the ALK gene: EML 4-ALK;
7) for the RET gene: NCOA4-RET, PRKAR1A-RET, PCM1-RET, KTN1-RET, KIF5B-RET, HOOK3-RET, GOLGA5-RET, CCDC 6-RET;
8) for the ROS1 gene: TPM3-ROS1, SLC34A2-ROS1, SDC4-ROS1, LRIG3-ROS1, GOPC-ROS1, EZR-ROS1, CD74-ROS 1;
9) for NTRK1/2/3 genes: TPM3-NTRK1, LMNA-NTRK1, NACC2-NTRK2, QKI-NTRK2, ETV6-NTRK 3.
In a second aspect, the present invention provides a gene chip for simultaneously detecting a plurality of gene mutation types, the gene chip comprising the probe set of the first aspect of the present invention.
In a third aspect, the present invention provides a method for detecting multiple types of gene mutations, the method comprising the steps of:
1) designing a probe: designing and synthesizing a probe set according to the first aspect of the invention;
2) DNA extraction and disruption: extracting DNA of a sample to be detected and breaking the DNA into DNA fragments of 200-250 bp;
3) library construction: constructing a sample library using the disrupted DNA;
4) and (3) hybridization and capture: contacting the sample library of step 3) with the probe set of step 1) for hybrid capture;
5) and (3) machine sequencing: sequencing the captured sequence obtained in step 4) to generate sequencing data;
6) information analysis: analyzing and annotating gene mutation information of the sequencing data obtained in the step 5).
In a fourth aspect, the present invention provides a kit for simultaneously detecting a plurality of gene mutation types, the kit comprising the probe set of the first aspect of the present invention or the gene chip of the second aspect of the present invention.
In a fifth aspect, the present invention provides the use of a probe set according to the first aspect of the present invention or a gene chip according to the second aspect of the present invention in the preparation of a detection reagent for simultaneously detecting one or more types of gene mutations.
The invention has the beneficial effects that:
(1) the invention can realize one-time simultaneous detection of gene mutations of 102 tumor driving genes including EGFR, KRAS, BRAF, c-MET, HER2, ALK, ROS1, RET, NTRK1/2/3 and the like, including point mutation, short fragment insertion deletion, copy number variation and gene fusion;
(2) the invention can detect the known mutation of the probe coverage area and can discover new unknown variation at the same time;
(3) the invention can detect related rare hotspot variation, namely 14 exon skipping mutation of MET gene, BCL2L11(BIM)2 intron deletion polymorphism related region and NTRK1/2/3 hotspot fusion mutation at one time;
(4) aiming at 6 genes-ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 of specific fusion breakpoint regions, the invention performs three times of high probe abundance coverage on the genes, so that the known hot spot fusion and unknown fusion variation of the genes can be detected more sensitively (0.5 percent);
(5) the invention is suitable for paraffin embedded tissues or paraffin sections, fresh pathological tissues, blood plasma, hydrothorax, ascites, cerebrospinal fluid and other sample types;
(6) the invention provides a detection method capable of simultaneously providing targeted medication and chemotherapy medication guidance.
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FIG. 1 is a flow chart of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The described embodiments are only a few embodiments of the invention, not all embodiments. All other embodiments are available to the person skilled in the art based on the embodiments of the invention and are within the scope of protection of the invention.
In the present specification and claims, when referring to a gene sequence, it will be understood by those skilled in the art that the gene sequence actually includes either or both of the complementary double strands. For convenience, in the present specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. For example, reference to a probe sequence actually includes the sequence and its complement. For example, reference is made to SEQ ID NO:1, actually including its complement. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.
The gene sequence in the present invention includes a DNA form or an RNA form, and in the case where one is disclosed, it means that the other is also disclosed. For example, reference to a probe sequence actually includes the corresponding RNA sequence.
As described above, there is a need in the art for a new technique that is comprehensive, highly sensitive, high-throughput, and simultaneously accurate for the detection of multiple mutation types.
The present invention provides a set of probes for simultaneously detecting multiple gene mutation types. The design length of the probe is 60-150nt, such as 120nt, and 100% coverage of a target area and at least more than 60% of high-specificity capture effect can be achieved through precise calculation. The designed probe is synthesized by the relevant synthesis companies such as IDT or Roche. A sample library is subjected to hybridization capture by adopting the enrichment probe designed by the invention, a corresponding sequencing platform is selected to carry out PE150 sequencing according to linker sequences (Illumina platform or Huada intelligent sequencing platform) of different sequencing platforms adopted during library construction, obtained sequencing data are obtained, then a single sample sequencing information analysis process is carried out, and effective mutation of a target medication related gene and a base type of a chemotherapy medication related gene are obtained, so that personalized medication guidance is carried out.
In the invention, the design of the probe aiming at the gene related to the targeted medication is based on databases such as TCGA, ICGC, COSMIC and the like and related literature references, and covers the comprehensive mutation sites of the hot tumor driving genes of common tumors such as lung cancer, colorectal cancer, gastric cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer, liver cancer and the like, including point mutation, short fragment insertion deletion, copy number variation and fusion genes, and the total of 5727 hot point mutations.
Accordingly, in a first aspect, the present invention provides a probe set for simultaneously detecting multiple gene mutation types, the probe set comprising a probe for a targeted medication-related gene, wherein the targeted medication-related gene comprises EGFR, BRAF, KRAS, HER2, MET, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3, and a gene mutation thereon comprises:
1) for the EGFR gene: g719, A698, V765, F712, L703, A702, G724, E709, S720, E709, S752_ I759del, L747_ S752del, L747_ T751del, E746_ A750del, E746_ S752> 747_ E749del, L747_ T751> 746_ E749del, K745_ E749del, L747_ T751del, S752_1759del, T751_ I759del, L747_ P753> 746_ S752del, E _ T751del, E746_ T751> 746_ T871, S752_ S747 _ T747 del, E _ T751> 746_ D779, G793D 793, G793D 79, G793D 770, GI 79858, GI 793, GI 779, GI 793, GI;
2) for the BRAF gene: a598_ T599insV, a598V, D594G, D594N, D594V, E586E, E586K, F583F, F595L, F595S, G596D, G596R, G606E, H608R, I582M, I592M, I592V, K601V, L584V, L597V, N593672, P213V, S605V, S V, T599_ V600insT, T599_ V600insTT, T599V, V600_ K601 593672 > V, V600, V36600, V600V V, V36600V V, V;
3) for the KRAS gene: g _ a11insG, G12G13insG, L19, G13, V14, G12, L19, G _ V14insG, G12, V8, a11, G12, G13, G15, a18, Q22, G13, a _ G12insGA, a59, Q61, T58, a146, K117;
4) for the HER2 gene: D769H, L755S, I767M, L755P, G776> VC, V777L, G776V, G778_ S779insG, V777A, V842I, H878Y;
5) for the MET gene: exon skipping mutations of E168D, T1010I, H1112Y, H1124D, H1112L, L1130L, N1118Y, H1112R, M1268T, Y1248C, Y1253D, 14;
6) for the ALK gene: EML 4-ALK;
7) for the RET gene: NCOA4-RET, PRKAR1A-RET, PCM1-RET, KTN1-RET, KIF5B-RET, HOOK3-RET, GOLGA5-RET, CCDC 6-RET;
8) for the ROS1 gene: TPM3-ROS1, SLC34A2-ROS1, SDC4-ROS1, LRIG3-ROS1, GOPC-ROS1, EZR-ROS1, CD74-ROS 1;
9) for NTRK1/2/3 genes: TPM3-NTRK1, LMNA-NTRK1, NACC2-NTRK2, QKI-NTRK2, ETV6-NTRK 3.
It is to be noted that in the present invention, the positions of the genes referred to above are based on the COSMIC database. However, one skilled in the art will appreciate that such genomic locations may correspond to locations of other versions of genomic data, as long as the relative locations of the genes correspond. In the present invention, mutations are expressed by a common expression in the art. For example, the mutations G719C and G719S indicate a mutation of glycine G at position 719 to cysteine C and serine S, respectively.
As will be appreciated by those skilled in the art, more accurate detection results can be obtained for a gene mutation with a relatively large number of probes. However, from the viewpoint of adverse interactions that may occur between the probes with each other and from the economical viewpoint, a typical combination of probes can be selected for these mutations, thereby achieving efficient detection.
Therefore, the inventors have screened the designed probes and found that the combination of some probes can realize effective detection. Thus, in one embodiment, the probe set comprises SEQ ID NO: 1-16.
In addition, for 6 genes of specific fusion breakpoint regions, namely ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3, the inventor carries out 3-fold high-abundance probe coverage on the genes, so that known hot spot fusion variation and unknown fusion variation can be detected more sensitively (0.5%).
Thus, in one embodiment, a probe set of the invention further comprises SEQ ID NO: 17-28.
SEQ ID NO:1-16 and SEQ ID NO: the sequences of 17-28 are shown in the following table.
In addition to the targeted drug-related genes described above, the present invention also relates to other targeted drug-related genes, aiming to more effectively screen out possible gene mutations and thus provide guidance for drug administration.
Thus, in one embodiment, the targeted drug-associated genes further comprise: AKT1, CDK4, FGFR2, MAP2K2, CDK6, FGFR3, PDGFRA, SMARCA4, APC, CDKN2A, FLT3, MLH1, PIK3CA, SMO, AR, CTNNB1, HRAS, MSH2, PMS2, SRC, ATM, DDR2, IDH1, MSH6, PTCH1, STK11, BCL2L11(BIM), IDH2, MTOR, PTEN, TP53, ERBB2, JAK2, NF1, PTPN11, TSC 11, BRCA 11, ESR 11, KIT, NRAS, RAF 11, TSC 11, BRCA 11, FBXW 11, RB, nd 11, VHL, cc3672, FGFR 11, MAP 2K.
The invention relates to a target medication related gene and a chemotherapy medication gene. Specifically, in one embodiment, the probe set further comprises a probe for a chemotherapy drug-related gene comprising: XRCC1, XPC, WT1, UMPS, UGT1A1, TYMS, TP53, STMN1, SOD2, SLIT1, SLC29A1, SLC28A3, SEMA3C, SELE, RRM1, PTGS2, NQO1, NLGN1, MTHFR, LTA, HTR1E, GSTP1, GSTO1, GGH, ESR2, ESR1, ERCC2, ERCC1, DYNC2H1, DPYD, DHFR, CYP3A4, CYP2D6, CYP19A1, COLEC10, CEP72, CDA, CBR3, C8orf34, AREG, ABCG 387 2, ABCC11, ABCB 1.
The design of the probe aiming at the chemotherapy drug related gene is based on databases such as Pharmgkb, Pubmed, FDA and the like and related documents and mechanisms, covers the current main chemotherapy drugs such as cisplatin, carboplatin, paclitaxel, docetaxel, vincristine, cyclophosphamide, epirubicin, other anthracyclines, gemcitabine, methotrexate, etoposide and the like, and detects 47 sites in total.
In a second aspect, the present invention provides a gene chip for simultaneously detecting a plurality of gene mutation types, the gene chip comprising the probe set of the first aspect of the present invention.
In a third aspect, the present invention provides a method for detecting multiple types of gene mutations, the method comprising the steps of:
1) designing a probe: designing and synthesizing a probe set according to the first aspect of the invention;
2) DNA extraction and disruption: extracting DNA of a sample to be detected and breaking the DNA into DNA fragments of 200-250 bp;
3) library construction: constructing a sample library using the disrupted DNA;
4) and (3) hybridization and capture: contacting the sample library of step 3) with the probe set of step 1) for hybrid capture;
5) and (3) machine sequencing: sequencing the captured sequence obtained in step 4) to generate sequencing data;
6) information analysis: analyzing and annotating gene mutation information of the sequencing data obtained in the step 5).
As described above, the probe design is based on databases such as TCGA, ICGC, COSMIC (for drug targeting related genes), Pharmgkb, Pubmed, FDA (for chemotherapy related genes), and related literature references. After the probe is designed, the relevant synthesis companies such as IDT or Roche are entrusted to complete the synthesis.
The DNA may be extracted from a suitable source. For example, in one embodiment, the extract can be extracted from paraffin-embedded tissue, paraffin sections, fresh pathological tissue, plasma, pleural fluid, ascites, cerebrospinal fluid, and other body fluid samples, but is not limited thereto. Any suitable sample may be used in the present invention.
The breaking of the DNA can be performed, for example, with an ultrasonic DNA breaking instrument. Thus, the DNA can be broken into 200-250bp DNA fragments.
It will be appreciated by those skilled in the art that one or more additional steps, such as fragment purification, library purification, amplification of captured products, etc., may be additionally introduced as desired in addition to the specific steps listed above.
In a fourth aspect, the present invention provides a kit for simultaneously detecting a plurality of gene mutation types, the kit comprising the probe set of the first aspect of the present invention or the gene chip of the second aspect of the present invention.
It will be appreciated that the kit may contain, in addition to the probe set or gene chip of the present invention, other reagents useful for detecting gene mutations.
For example, in one embodiment, the kit may further comprise: (1) DNA extraction reagents, such as reagents suitable for tumor cfDNA, FFPE tissue extraction; (2) a library-building agent; (3) primers and linkers, depending on the requirements of the sequencing platform, such as the Illumina platform or the cissus sequencing platform; (4) a nucleic acid purification reagent; (5) positive quality control material and negative quality control material.
The kit can effectively meet the individual medication guidance requirement of the tumor.
In a fifth aspect, the present invention provides the use of a probe set according to the first aspect of the present invention or a gene chip according to the second aspect of the present invention in the preparation of a detection reagent for simultaneously detecting one or more types of gene mutations.
As described above, the present invention can detect mutations in up to 102 genes at a time, thereby finding known and/or unknown mutations; the invention can detect related rare hotspot variation, namely 14 exon skipping mutation of MET gene, BCL2L11(BIM)2 intron deletion polymorphism related region and NTRK1/2/3 hotspot fusion mutation at one time; because the abundance coverage of the probes is carried out three times for the genes ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 of 6 specific fusion breakpoint regions, the invention can detect the known hot spot fusion and unknown fusion variation thereof more sensitively (0.5 percent).
The invention will now be further described by way of the following examples. The test methods used in the following examples are conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
DNA extraction and disruption
Taking 10-20 Tissue slices of 1 test paraffin-embedded case Tissue, extracting DNA of the paraffin-embedded case Tissue sample according to the instruction of a QIAamp DNA FFPE Tissue Kit (Qiagen) extraction Kit, and then quantifying by using the Qubit, wherein the Tissue DNA is required to be at least more than 100 ng. And breaking the extracted tissue DNA into DNA fragments of 200bp-250bp by using an ultrasonic DNA breaking instrument, and constructing a sample library according to a library construction kit.
2. Library construction
2.1 end repair/dA Tail addition
In a sterile 1.5ml PCR tube, the end repair and addition "a" reactions were performed according to the following table configuration:
gently pipetting or shaking the mixture by using a pipettor, centrifuging the reaction solution to the bottom of the tube by short-time centrifugation, and then putting the tube into a PCR instrument for processing according to the following procedures: 20 minutes at 30 ℃ and 20 minutes at 70 ℃.
2.2 Joint connection
Based on the product of the previous step, a specific MGISEQ2000 linker was attached according to the following table configuration.
Gently pipetting or shaking the mixture by using a pipettor, centrifuging the reaction solution to the bottom of the tube by short-time centrifugation, and then putting the tube into a PCR instrument for processing according to the following procedures: 15 minutes at 20 ℃.
2.3 magnetic bead purification of ligation products
And (3) carrying out fragment sorting and purification on the product obtained in the step 2.2, wherein the specific operation steps are as follows:
1) preparation work: taking the DNA Selection Beads magnetic Beads out of the refrigerator, and balancing at room temperature for at least 30 minutes; preparing 80% ethanol.
2) The beads were mixed with 80% ethanol and vortexed or the beads were inverted sufficiently to ensure adequate mixing.
3) 60 μ L of DNA Selection Beads (0.6X, Beads: DNA ═ 0.6: 1) were pipetted into the adaptor ligation and incubated for 5 min at room temperature.
4) The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads and liquid, and after the solution cleared (about 5 minutes), the supernatant was carefully removed.
5) The PCR tube was kept in the magnetic rack all the time, 200. mu.L of freshly prepared 80% ethanol was added to rinse the beads, and after incubation at room temperature for 30 seconds, the supernatant was carefully removed.
6) Repeat step 5) for a total of two rinses.
7) The PCR tube was kept in the magnetic stand all the time, and the beads were air dried with the lid open until cracking had just occurred (no more than 5 minutes).
8) The PCR tube was removed from the magnetic stand and 21. mu.L ddH was added2And O, vortexing or gently beating by using a pipette until the mixture is fully mixed, and standing for 5 minutes at room temperature.
9) The PCR tube was briefly centrifuged and placed in a magnetic stand and left to stand, after the solution cleared (about 5 minutes), 20. mu.L of the supernatant was carefully removed to a new PCR tube, and the beads were removed.
2.4 library amplification and purification
In a sterile 0.2ml PCR tube, a PCR reaction system is configured according to the following table, and PCR amplification enrichment is carried out on the joint connection product.
Gently blowing or shaking and mixing uniformly by using a pipettor, centrifuging the reaction solution for a short time to the bottom of the tube, and then putting the reaction solution into a PCR instrument for PCR, wherein the PCR reaction conditions are as follows:
the amplified product was subjected to fragment sorting and purification using DNA Selection Beads (0.9 ×, Beads: DNA ═ 0.9: 1). The purified product needs to be subjected to Qubit-BR quantification and 2100 quality control. Generally, a qualified library product requires a concentration greater than 20 ng/. mu.L, and a major peak of the target fragment of about 380 bp.
3. Enrichment of target sequences and sequencing on machine
3.1 after the quality control of the amplified library is qualified, hybridization capture is performed by using the probe set designed by the invention, referring to the instruction provided by the chip manufacturer (IDT). Finally, elution and redissolution of 21. mu.L ddH2And (4) hybridizing an O band to elute the magnetic beads.
3.2 amplification of hybrid Capture products
3.3 removing the magnetic beads in the previous step, then purifying the magnetic beads, and finally redissolving 25 μ L of ddH2And O, performing QC and operation.
3.4 performing on-machine sequencing on PE150 by using a MGISEQ2000 sequencer, and performing on-machine sequencing on sequencing experiment operation according to an operation instruction provided by a manufacturer.
4. Information analysis and result sorting
And after the data is downloaded, splitting a detection sample based on the barcode sequence, and simultaneously deleting low-quality data. The specific parameters are as follows: filtering out fragments with sequence head-to-tail quality values below 20; discarding short sequences lower than 40 bp; filtering the N base fragment and the adaptor sequence fragment with the ratio of more than 30 percent, and the like.
According to the position form (UID) of the template fragment, performing clustering marking and replication, simultaneously correcting and modifying the base quality value of an error base introduced by sequencing through the clustering condition, then performing re-alignment by using BWAmen, and performing alignment and replication by using GATK.
Carrying out call homogeneous SNV mutation by using mutect, varScan and somVar processes; performing variation by using gatk, varScan and somVar flow call homogeneous InDel; call CNV mutation is carried out by using a contra. py procedure; finally annotating the function of variation, the number of reads supported, the variation frequency, the amino acid variation, variation labels in Cosmic and the like by using the call SV in the somVar process.
The obtained variation result needs to be filtered based on relevant conditions so as to obtain the finally meaningful relevant mutation, and specific criteria are as follows: a. filtering out mutations that are not in the exon (exonic) region, splicing (splicing) region, exon and splicing region; b. filtering out synonymous mutations; c. filtering out variations with quality values below 30 in the repeat region; d. polymorphic mutations greater than 0.01 in the 1000G, exac, esp6500 database were filtered out.
5. And (3) analyzing a detection result:
5.1 Probe Performance evaluation (QC):
typically, a 30% coverage X of greater than 90% indicates good coverage. The detection result of the invention reaches 96.07 percent, which shows that the target interval of the detection is effectively covered.
Generally, the higher the capture efficiency of the probe, the better the specificity of the probe, and the more effective the cost of sequencing can be reduced. The capture efficiency of the invention reaches 65.47%, which is 50% higher than that of most organizations in the industry. This indicates good probe specificity.
5.2 mutation detection results
The results of detecting 3 mutations of the target genes are completely consistent with the results of known mutation types and frequencies, the details are shown in table 3, and the chemotherapy 47-site base types are also completely consistent with the known base types.
TABLE 3 details of the results of the Targeted Gene variations
5.3 interpretation of results
Based on the variation of the target gene EGFR L858R & E746A 750del, the patient is predicted to be sensitive to EGFR-TKIs medicaments, such as erlotinib, oxitinib, dacomitinib, gefitinib, erlotinib and afatinib. Based on the ALK fusion mutation of the target gene, the patient can be expected to use ALK inhibitors such as crizotinib, idetinib, ceritinib, bugatitinib and loratinib.
Based on the information of 47 chemotherapy detection sites, the calculation shows that the patients have better curative effects on the pyrimidine analogues of capecitabine, fluorouracil, tegafur, gemcitabine and raltitrexed, but the specific medication also needs to be combined with clinical caution.
Sequence listing
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Claims (10)
1. A probe set for simultaneously detecting multiple gene mutation types, the probe set comprising probes for targeted medication-related genes, wherein the targeted medication-related genes comprise EGFR, BRAF, KRAS, HER2, MET, ALK, RET, ROS1, NTRK1, NTRK2, NTRK3, and gene mutations thereon comprise:
1) for the EGFR gene: g719, A698, V765, F712, L703, A702, G724, E709, S720, E709, S752_ I759del, L747_ S752del, L747_ T751del, E746_ A750del, E746_ S752> 747_ E749del, L747_ T751> 746_ E749del, K745_ E749del, L747_ T751del, S752_ I759del, T751_ I759del, L747_ P753> 746_ S752del, E _ T751del, E746_ T871 > 871 _ S752> 747_ T751> 747_ A750> 747_ P753> 797 _ T774, E _ D793, G793D 793, D79, D769, D770, GI 793, 793D 793, D793D 769, D770, 3D 79, 3D 793, 3D 793, 3D 7944, 3D 793, 3D 7944, 3D 770, 3D 7944, 3D 7944, 3D 7678, 3D 7944, 3D 793, 3D 79;
2) for the BRAF gene: a598_ T599insV, a598V, D594G, D594N, D594V, E586E, E586K, F583F, F595L, F595S, G596D, G596R, G606E, H608R, I582M, I592M, I592V, K601V, L584V, L597V, N593672, P213V, S605V, S V, T599_ V600insT, T599_ V600insTT, T599V, V600_ K601> V, V600, V V, V600V V, V36600V V, V;
3) for the KRAS gene: g _ a11insG, G _ G13insG, L19, G13, V14, G12, L19, G _ V14insG, G12, V8, a11, G12, G13, G15, a18, Q22, G13, a _ G12insGA, a59, Q61, T58, a146, K117;
4) for the HER2 gene: D769H, L755S, I767M, L755P, G776> VC, V777L, G776V, G778_ S779insG, V777A, V842I, H878Y;
5) for the MET gene: exon skipping mutations of E168D, T1010I, H1112Y, H1124D, H1112L, L1130L, N1118Y, H1112R, M1268T, Y1248C, Y1253D, 14;
6) for the ALK gene: EML 4-ALK;
7) for the RET gene: NCOA4-RET, PRKAR1A-RET, PCM1-RET, KTN1-RET, KIF5B-RET, HOOK3-RET, GOLGA5-RET, CCDC 6-RET;
8) for the ROS1 gene: TPM3-ROS1, SLC34A2-ROS1, SDC4-ROS1, LRIG3-ROS1, GOPC-ROS1, EZR-ROS1, CD74-ROS 1;
9) for NTRK1/2/3 genes: TPM3-NTRK1, LMNA-NTRK1, NACC2-NTRK2, QKI-NTRK2, ETV6-NTRK 3.
2. The probe set of claim 1, comprising the probe sequences set forth in SEQ ID NOS 1-16.
3. The probe set according to claim 1 or 2, further comprising the probe sequences shown in SEQ ID NO 17-28.
4. The panel of claim 1-3, wherein said targeted drug-associated gene further comprises: AKT1, CDK4, FGFR2, MAP2K2, CDK6, FGFR3, PDGFRA, SMARCA4, APC, CDKN2A, FLT3, MLH1, PIK3CA, SMO, AR, CTNNB1, HRAS, MSH2, PMS2, SRC, ATM, DDR2, IDH1, MSH6, PTCH1, STK11, BCL2L11(BIM), IDH2, MTOR, PTEN, TP53, ERBB2, JAK2, NF1, PTPN11, TSC 11, BRCA 11, ESR 11, KIT, NRAS, RAF 11, TSC 11, BRCA 11, FBXW 11, RB, nd 11, VHL, cc3672, FGFR 11, MAP 2K.
5. The set of probes of any of claims 1-4, wherein said set of probes further comprises probes for chemotherapeutic drug-related genes comprising:
XRCC1、XPC、WT1、UMPS、UGT1A1、TYMS、TP53、STMN1、SOD2、SLIT1、SLC29A1、SLC28A3、SEMA3C、SELE、RRM1、PTGS2、NQO1、NLGN1、MTHFR、LTA、HTR1E、GSTP1、GSTO1、GGH、ESR2、ESR1、ERCC2、ERCC1、DYNC2H1、DPYD、DHFR、CYP3A4、CYP2D6、CYP19A1、COLEC10、CEP72、CDA、CBR3、C8orf34、AREG、ABCG2、ABCC11、ABCB1。
6. a gene chip for simultaneously detecting a plurality of gene mutation types, the gene chip comprising the probe set of any one of claims 1 to 5.
7. A method for detecting multiple gene mutation types, the method comprising the steps of:
1) designing a probe: designing and synthesizing a set of probes according to any one of claims 1-5;
2) DNA extraction and disruption: extracting DNA of a sample to be detected and breaking the DNA into DNA fragments of 200-250 bp;
3) library construction: constructing a sample library using the disrupted DNA;
4) and (3) hybridization and capture: contacting the sample library of step 3) with the probe set of step 1) for hybrid capture;
5) and (3) machine sequencing: sequencing the captured sequence obtained in step 4) to generate sequencing data;
6) information analysis: analyzing and annotating gene mutation information of the sequencing data obtained in the step 5).
8. The method of claim 7, wherein the test sample comprises a body fluid sample such as paraffin-embedded tissue, paraffin sections, fresh pathological tissue, plasma, pleural fluid, ascites, cerebrospinal fluid, and the like.
9. A kit for simultaneously detecting a plurality of gene mutation types, the kit comprising the probe set of any one of claims 1 to 5 or the gene chip of claim 6.
10. Use of a set of probes according to any one of claims 1 to 5 or a gene chip according to claim 6 for the preparation of a detection reagent for the simultaneous detection of one or more types of genetic mutations.
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