CN111187810A - Method for detecting multiple tumor-associated genes for non-diagnostic therapeutic purposes - Google Patents

Abstract

The invention provides a method for detecting a plurality of tumor-associated genes for non-diagnostic therapeutic purposes, which comprises (a) extracting DNA from a sample; constructing a DNA library based on the DNA of step (a); hybridizing the DNA library with a specially-made probe library, and purifying the hybridized library, wherein the special probe library at least comprises a plurality of capture probes designed aiming at a gene where a mononucleotide polymorphic chain related to a chemotherapeutic drug is located, a targeted drug related gene, a gene related to the stability of a microsatellite for immunotherapy and a virus gene; (d) sequencing the DNA result obtained in step (c) and detecting the genetic variation. In the invention, the multi-gene enriched probe library can enrich and detect a plurality of cancer-related genes at one time, provide related biological information and provide references of various clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy for various cancer patients.

Description

Method for detecting multiple tumor-associated genes for non-diagnostic therapeutic purposes

The application is filed as24 days 12 months in 2018Application No. is201811584036.3The invention is named asMultiple bases Method for detecting multiple genes related to multiple tumor treatments due to enriched probe libraryDivisional application of the patent application.

Technical Field

The invention belongs to the field of gene detection, and particularly relates to a method for detecting multiple genes related to multiple tumors for non-diagnosis and treatment purposes.

Background

Tumors are one of the diseases seriously harming human health, and are one of the leading causes of human death. In china, 1/4 deaths died from tumors, beginning as the cause of death. According to 2 months in 2017, the latest Chinese cancer data issued by the national cancer center shows that 368 thousands of new Chinese cases account for 1/4 of new cancer cases in the world in 2013, the incidence rate is 186/10 thousands of people, and the mortality rate is 109/10 thousands of people.

The selection of the correct treatment modality and drug is critical for the treatment of tumors. The National Comprehensive Cancer Network (NCCN) promulgates various clinical practice guidelines for malignancy every year, which are recognized and followed by clinicians worldwide. The treatment means of cancer mainly comprises traditional treatment means such as surgery, radiotherapy, chemotherapy, traditional Chinese medicine treatment and the like, and latest treatment means such as targeted treatment, immunotherapy and the like. The targeted therapy is to design a corresponding therapeutic drug aiming at a well-defined carcinogenic site (the site can be a protein molecule inside a tumor cell or a gene segment), and the drug enters into the body to be specifically combined with the carcinogenic site to take effect, so that the tumor cell is specifically killed without affecting normal tissue cells around the tumor. Immunotherapy (immunotherapy) refers to a therapeutic method for artificially enhancing or suppressing the immune function of the body to treat diseases in response to a low or high immune state of the body. Whether targeted or immunotherapy, the choice of treatment regimen and prognosis are significantly linked to the presence of genetic variation in tumor patients. As indicated by the 2018 release 1 colon cancer treatment guidelines issued by NCCN, patients had known KRAS mutations (exons 2,3,4) or NRAS mutations (exons 2,3,4) that were not effective against cetuximab and panitumumab. The immunotherapeutic drug Keytruda (pamumab), which can be used to treat adult and pediatric patients with unresectable or metastatic solid tumors with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR), is the first non-compliant tumor source approved by the U.S. Food and Drug Administration (FDA) but an anti-tumor therapy that is differentiated according to biomarkers. In addition to the latest tumor treatment methods such as targeted therapy and immunotherapy, the genetic variation of tumor patients also affects the treatment effect of chemotherapy. The Single Nucleotide Polymorphism (SNP) state of a cancer patient has a certain reference value for judging the effect of a chemotherapeutic drug and the degree of toxic and side effects, such as SNP in ERCC1 gene, and the sensitivity of a C/C genotype patient to platinum chemotherapy is higher than that of C/T or T/T type. Therefore, the detection of the gene variation condition of the tumor patient is beneficial to realizing accurate treatment of the tumor.

Next-generation sequencing (NGS), also known as high-throughput sequencing, can perform sequence determination on hundreds of thousands to millions of nucleic acid molecules at a time, and can synchronously obtain all sequence information in individual samples of tumor patients. The genetic variation condition on the molecular level of a tumor patient can be known more quickly and accurately by utilizing the second-generation sequencing technology, so that accurate treatment is carried out. With the advantages of the precise treatment in tumor treatment, the development of a multigene enrichment and detection method for pan-cancer clinical treatment guidance based on the second-generation sequencing technology is particularly important. The method can judge the genetic variation condition of the patient through one-time detection, provide related biological information, provide references of various clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy for various cancer patients, maximize the treatment effect, prolong the life cycle of the patient and improve the life quality of the patient.

Disclosure of Invention

The present disclosure has been made in view of the above-mentioned problems of the prior art, and an object of the present disclosure is to provide a multi-gene enrichment probe library and applications thereof, which can provide biological information related to genetic variation of a patient by only one-time detection, and provide reference information for a course management protocol of a plurality of cancer patients.

To this end, the present disclosure provides a method for detecting a plurality of tumor-associated genes for non-diagnostic therapeutic purposes, which may comprise: (a) extracting DNA from the sample; (b) constructing a DNA library based on the DNA of step (a); (c) hybridizing the DNA library with a specially-made probe library and purifying the hybridized library, wherein the specially-made probe library comprises a plurality of capture probes designed aiming at a plurality of genes related to a plurality of tumor treatments, the 3 'ends of the plurality of capture probes are provided with biotin marks, the biotin marks are arranged at any position from 1 st to 5 th positions of the reciprocal of the 3' end, the plurality of genes at least comprise genes of mononucleotide polymorphic chains related to chemotherapeutic drugs, targeted drug related genes, genes related to the stability of microsatellites for immunotherapy and genes of viruses capable of causing cancers, the genes related to the stability of the microsatellites for immunotherapy are used for judging tumor related labels comprising tumor mutation loads, and the genes of the viruses comprise EBV viruses related to nasopharyngeal carcinogenesis, HPV virus related to the occurrence of cervical carcinoma and HBV and HCV virus related to the occurrence of liver cancer, wherein the plurality of capture probes comprise: 1-19 of the sequence SEQ ID NO; probes designed for microsatellite stability-related genes for immunotherapy include SEQ ID NOs 11-15; probes designed for viral genes include SEQ ID NO 16-19; and (d) sequencing the DNA result obtained in step (c) to detect genetic variation.

According to the method for detecting the multiple tumor-associated genes for non-diagnostic treatment purposes, provided by the disclosure, the multiple cancer-associated genes can be enriched and detected at one time, relevant biological information including whether the viral oncogenes are carried in the patient body is provided, and references of multiple clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy are provided for the multiple cancer patients. In addition, since the 3 'ends of the plurality of capture probes have the biotin label and the biotin label may be located at any one of the 1 st to 5 th positions of the reciprocal of the 3' ends, it is possible to easily purify DNA and reduce the influence of the biotin label on the probes.

In addition, in the detection method according to the present disclosure, optionally, in the step (b), the DNA extracted from the step (a) may be fragmented, and the fragmented DNA fragments are subjected to end repair and a-tailing, ligated with a sequencing adaptor and purified, thereby performing PCR amplification to obtain the DNA library. Thus, a desired DNA library can be obtained.

In addition, in the detection method according to the present disclosure, the sample may be a human-derived sample. This method can be applied to the detection of human genes.

In addition, in the detection method according to the present disclosure, optionally, in step (c), when the purification is performed: uniformly mixing the avidin-treated magnetic beads with the hybridized library to form a sample to be purified, and standing; and (3) placing the sample to be purified on a magnetic rack for magnetic bead adsorption until the solution is clarified, washing the magnetic beads by using ethanol, and then drying the magnetic beads at room temperature. In this case, avidin can specifically bind to biotin, thereby achieving purification.

In addition, in the detection method related to the present disclosure, the genetic variation may optionally include at least one of a point mutation, a gene fragment insertion/deletion, a copy number change, and a fusion/gene rearrangement. This enables more comprehensive detection of gene mutation.

In addition, in the detection method according to the present disclosure, optionally, in the step (c), the plurality of capture probes have a length of 50 to 300bp, and cover a target region and flanking sequences on both sides of the target region, the target region being DNA sequences of the plurality of genes, and the flanking sequences being sequences in a range of 5 to 50bp on both ends of the DNA sequences; and the plurality of capture probes are designed in a shingled manner, and each of the plurality of capture probes has an overlap portion between them, the overlap portion being 5 to 20 bases. In this case, the flanking sequence is favorable for improving the specificity and accuracy of the DNA probe, the length of the capture probe can be suitable for the length of the gene to be captured, and the shingle design among the probes can improve the coverage of the capture probe, so that the probes are favorable for better capturing the gene and detecting the gene variation.

In addition, in the detection method according to the present disclosure, optionally, in the step (d), data analysis may be performed on the result of the sequencing to detect genetic variation, the data analysis including removing unsatisfactory data on the result of the sequencing, the unsatisfactory data including data having mapping quality (mapping quality) of Q10 or less and polyN data of 20 or more consecutive identical bases, and clipping the data result after the removal. Therefore, more accurate biological information can be obtained.

In addition, in the detection method according to the present disclosure, optionally, in the step (b), the DNA extracted from the step (a) may be fragmented using a fragmentation enzyme or an ultrasonicator. Thus, a large DNA fragment can be easily fragmented to obtain a small DNA fragment.

In addition, in the detection method according to the present disclosure, optionally, in the step (b), after the DNA fragment is connected to the sequencing adaptor, magnetic bead adsorption may be used for double screening, and the double screening step includes: adding a specified amount of buffer solution into a sample tube containing the DNA fragments, uniformly mixing by vortex, standing at room temperature, and adsorbing the DNA fragments by using magnetic beads until the solution is clear; and taking out the supernatant from the sample tube, uniformly mixing the supernatant with the supernatant in a vortex manner, standing at room temperature, and adding the magnetic beads again to adsorb the DNA fragments. Thus, the prepared DNA library can be sufficiently collected.

In the detection method according to the present disclosure, optionally, in step (c), the length of the plurality of capture probes may be 120bp, the flanking sequences may be sequences within 10bp of both ends of the DNA sequence, and the overlapping portion may be 12 bases. Therefore, the method is beneficial to better capture genes and detect gene variation by the probe.

According to the present disclosure, a plurality of cancer-related genes can be enriched and detected at one time, relevant biological information including whether a patient carries a viral oncogene or not is provided, and references of various clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy are provided for a plurality of cancer patients.

It should be noted that the detection method of tumor genes according to the present application, the detection result thereof, is only used for analyzing and judging the biological information of the relevant genes, and provides reliable analysis basis for drug administration or treatment.

Drawings

Fig. 1 is a flowchart showing a method for detecting a plurality of genes related to a plurality of tumors according to an embodiment of the present invention.

FIG. 2 is a flowchart showing the construction of a DNA library according to an embodiment of the present invention.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same components are denoted by the same reference numerals, and redundant description thereof is omitted. The drawings are schematic and the ratio of the dimensions of the components and the shapes of the components may be different from the actual ones.

The multi-gene enriched probe library of the present disclosure may include a plurality of capture probes designed for a plurality of genes associated with a plurality of tumors, the plurality of genes including at least a gene in which a single nucleotide polymorphism associated with a chemotherapeutic drug is located, a targeted drug-related gene, and a microsatellite stability-related gene and a viral gene for immunotherapy. In this case, the multi-gene enriched probe library can enrich and detect a plurality of cancer-related genes at one time, can provide relevant biological information including whether a patient carries a virus oncogene, and provides a reference for a plurality of clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy for a plurality of cancer patients. In addition, since the 3 'ends of the plurality of capture probes have the biotin label and the biotin label may be located at any one of the 1 st to 5 th positions of the reciprocal of the 3' ends, it is possible to easily purify DNA and reduce the influence of the biotin label on the probes.

In this embodiment, among the plurality of genes related to tumor treatment, a gene in which a Single Nucleotide Polymorphism (SNP) related to a chemotherapeutic drug is located can be used to provide a reference for determining sensitivity and toxic and side effects of the chemotherapeutic drug, thereby evaluating a chemotherapeutic regimen. In addition, genes associated with targeted drugs can be used for selection of targeted drugs and to evaluate the efficacy of targeted therapies. In addition, the gene related to the stability of the microsatellite can be used for providing a reference for judging the effect of the immunotherapy medicament and can also be used for judging the Tumor mutation load (TMB) of a Tumor patient, namely the total number of somatic gene coding errors, base substitution, gene insertion or deletion errors detected in each million bases. In addition, the virus-associated genes can be used to determine whether a tumor is associated with a viral infection, thereby providing a physician with reference to the selection of an appropriate treatment regimen.

In some examples, preferably, the plurality of genes associated with the tumor is no less than 200; more preferably, the number of tumor-associated genes is not less than 400; further preferably, the number of tumor-associated genes is not less than 600. In addition, in some examples, the number of tumor-associated genes is 600 to 800.

In addition, in some examples, probes designed for the gene in which the single nucleotide polymorphic strand associated with a chemotherapeutic drug is located can include SEQ ID NO 1-SEQ ID NO 5. Probes designed for genes associated with targeted drugs may include SEQ ID NO 6-SEQ ID NO 10. Probes designed for microsatellite stability-related genes for immunotherapy may include SEQ ID NO 11-SEQ ID NO 15. Probes designed for viral genes may include SEQ ID NO 16-SEQ ID NO 19.

Additionally, in some examples, the viral gene may be a gene of a virus capable of causing cancer. In this case, the multi-gene enriched probe library can enrich and detect viral genes associated with cancer, and further determine whether a tumor is associated with viral infection, thereby providing a reference for a physician to select an appropriate treatment regimen. For example, in some examples, the viral gene may be at least one of EBV virus associated with nasopharyngeal carcinogenesis, HPV virus associated with cervical carcinogenesis, HBV associated with hepatoma carcinogenesis, and HCV virus.

In addition, in this embodiment, the capture probes in the probe library may include: the sequence SEQ ID NO. 1-SEQ ID NO. 19. Therefore, genes related to cancer treatment can be captured, so that the disease course management scheme evaluation can be carried out, and a reference can be provided for clinical treatment.

In this embodiment, the 3' ends of the plurality of capture probes may be labeled with biotin. Biotin and avidin bind rapidly and specifically, and thus, DNA can be purified easily.

In this embodiment, biotin may be labeled at least any one of positions 1 to 5 from the 3' -end. In this case, the influence of the biotin label on the probe can be reduced. For example, in one example, biotin can be labeled at the 2 nd position from the 3' terminus. In another example, biotin can be labeled at the 4 th position from the 3' terminus. In addition, in yet another example, biotin may be labeled at the 3 rd position from the 3' terminus.

In this embodiment, a DNA probe that hybridizes to a target region may be included, and the target region may be a DNA sequence of a plurality of genes and flanking sequences on both sides of the DNA sequence. In this case, the specificity and accuracy of the DNA probe can be improved.

In some examples, each probe of the plurality of capture probes can be between 50bp and 300bp in length. In this case, the length of the capture probes is appropriate to the length of the gene to be captured, which is beneficial for the probes to capture the gene better. For example, in one example, each probe of the plurality of capture probes can be 50bp in length. In another example, each probe of the plurality of capture probes can be 200bp in length. In addition, in yet another example, the length of the probe may be 300 bp.

In some examples, the flanking sequences may be sequences ranging from 5bp to 30bp on either end of the DNA sequence. In this case, the specificity of the capture probe can be improved. For example, in one example, the flanking sequences may be sequences that range from 5bp on either side of the DNA sequence. In another example, the flanking sequences may be sequences that are 20bp apart from the DNA sequence. In addition, in yet another example, the flanking sequences may be sequences that range from 30bp on either side of the DNA sequence.

In addition, in the present embodiment, the plurality of capture probes may be designed in a shingled manner with an overlap between each probe of the plurality of capture probes. This improves the coverage of the capture probes and allows efficient detection of genetic variations.

In some examples, the overlap between each probe of the plurality of capture probes can be 5 to 20 bases. This overlap of ranges does not adversely affect the capture of the gene by the probe. For example, in one example, the overlap between each of the plurality of capture probes can be 5 bases. In another example, the overlap between each of the plurality of capture probes can be 15 bases. In addition, in yet another example, the overlap between each of the plurality of capture probes can be 20 bases.

Hereinafter, a method for detecting a plurality of genes associated with a plurality of tumors will be described in detail with reference to fig. 1 and 2. Fig. 1 is a flowchart showing a method for detecting a plurality of genes related to a plurality of tumors according to an embodiment of the present invention. FIG. 2 is a flowchart showing the construction of a DNA library according to an embodiment of the present invention.

In the method for detecting a plurality of genes associated with a plurality of tumors according to the present disclosure, the following steps may be included: extracting DNA from the sample (step S10); constructing a DNA library based on the DNA obtained in step S10 (step S20); hybridizing the DNA library with the multigene-enriched probe pool of the present disclosure, and purifying the hybridized library (step S30); the DNA result obtained in step S30 is sequenced to detect a genetic variation (step S40).

In addition, in the present embodiment, in step S20, the DNA extracted from step S10 is fragmented (step S21), and the fragmented DNA fragments are subjected to end repair and a-tailing (step S22), followed by ligation and purification of a sequencing adaptor (step S23), and then PCR amplification is performed to obtain a DNA library (step S24). Thus, a desired DNA library can be obtained.

In some examples, the sample from which DNA is extracted may be a human sample. Alternatively, the human sample may include whole genomic DNA extracted from tissues or cells such as biopsy tissue, paraffin-embedded tissue, and blood cells, or free DNA extracted from body fluids, blood. In some examples, if the sample from which the DNA is extracted in step S10 is a body fluid, blood-extracted free DNA, step S21 may not be required, i.e., the extracted DNA does not need to be fragmented.

In addition, in some examples, in step S21, DNA fragmentation may be performed by standard means in the art, for example, DNA fragmentation may be performed by a physical method such as a sonicator, or DNA fragmentation may be performed by a biological method such as a fragmenting enzyme. In the present embodiment, it is preferable to perform DNA fragmentation using a fragmenting enzyme. Thus, a large DNA fragment can be easily fragmented to obtain a small DNA fragment.

In this embodiment, in step S20, after the DNA fragments are ligated to the sequencing adaptors, double-screening may be performed by magnetic bead adsorption, and the double-screening step may include: adding a specified amount of buffer solution into a sample tube containing the DNA fragments, uniformly mixing by vortex, standing at room temperature, and adsorbing the DNA fragments by using magnetic beads until the solution is clear; and taking out the supernatant from the sample tube, uniformly mixing the supernatant with the supernatant in a vortex manner, standing at room temperature, and adding the magnetic beads again to adsorb the DNA fragments. Thus, the prepared DNA library can be sufficiently collected.

In this embodiment, in step S30, the library after hybridization may be purified using an avidin-treated magnetic bead. In this case, the purification efficiency can be improved by utilizing the property of rapid and specific binding of avidin to a label such as biotin. In some examples, the avidin may be at least one of streptavidin, ovalbumin. In some examples, the avidin is preferably streptavidin.

In addition, in the present embodiment, in the step S30, when the purification is performed: uniformly mixing the avidin-treated magnetic beads with the hybridized library to form a sample to be purified, and standing; and (3) placing the sample to be purified on a magnetic rack for magnetic bead adsorption until the solution is clarified, washing the magnetic beads by using ethanol, and then drying the magnetic beads at room temperature. In this case, avidin can specifically bind to biotin, thereby achieving purification.

In addition, in some examples, the genetic variation includes at least one of a point mutation, a gene fragment insertion/deletion, a copy number change, and a fusion/gene rearrangement genetic variation. This enables more comprehensive detection of gene mutation.

In addition, in this embodiment, in step S40, a tumor-associated signature including at least microsatellite instability and tumor burden mutations may also be detected.

In this embodiment, in step S40, data analysis is performed on the sequencing result to detect a gene mutation. In this case, relevant biological information can be provided, providing reference to a variety of clinical treatment course management protocols including chemotherapy, targeted therapy and immunotherapy for a variety of cancer patients.

In addition, in this embodiment, the data analysis may include removing the non-conforming data from the results of the sequencing and clipping the data results after removal. Preferably, the cropping removes the last 8-10nt of reads. In this case, the final accuracy of the sequencing result can be ensured.

In some examples, the failure data may include data with a genome mapping (mapping) quality less than or equal to Q10. In this case, the data that has generated the error can be eliminated, and the accuracy of the result can be further improved.

In some examples, the fail data may also include polyN data for more than 20 consecutive identical bases. In this case, the data that has generated the error can be eliminated, and the accuracy of the result can be further improved.

As described above, in the present embodiment, a multi-gene enriched probe library and applications thereof can be provided, and the probe library can provide biological information of a plurality of tumor-associated genes through only one detection, and provide references of a plurality of clinical treatment course management schemes including chemotherapy, targeted therapy and immunotherapy for a plurality of cancer patients. .

Hereinafter, embodiments of the present invention will be described in further detail with reference to specific examples.

[ example 1 ]

In this example, probe design and synthesis were performed using probe design software:

(1) screening of tumor treatment related genes: the method integrates the drug information approved by FDA in the United states, the clinical practice guideline for malignant tumors published by NCCN in the United states, Cancer-related Somatic Mutations covered by Cancer Somatic mutation Catalogue (Catalogue of viral Mutations in Cancer, COSMIC), the association between genes and drugs and the association information of gene SNP and chemotherapeutic drugs provided by PharmGKB database, and the literature and reports aiming at Cancer treatment, integrates and screens the genes related to Cancer treatment, and obtains a total of 605 genes.

(2) Probes were designed using Primer Premier 5.0, based on the human whole genome sequence published by the National Center for Biotechnology Information (NCBI) of the United states as a reference; designing a plurality of DNA probes capable of hybridizing with a target region by taking a DNA sequence of the target gene and flanking sequences at two sides of the DNA sequence as the target region; the length of the DNA probe is limited to 120 bases, the probe is designed in a shingled manner and completely covers all the exons of the gene to be detected and sequences within the range of 10bp at two ends, and the overlapping part between the probes is 12 bases.

(3) Probes designed according to 605 genes are obtained, and partial probes are shown as SEQ ID NO. 1-SEQ ID NO. 19.

(4) And (3) probe synthesis: probes were synthesized by Roche diagnostics products Ltd.

[ example 2 ]

This example utilizes a pool of multi-gene enriched probes for hybrid capture, followed by sequencing of the captured sequences.

(a) Extraction of DNA from the sample: DNA extraction of all samples can be performed by any standard means known in the art. The sample DNA in this example was a puncture paraffin section sample HP201701 of 1 breast cancer patient, and was extracted using a GeneRead DNA FFPE Kit from QIAGEN, Germany.

(b) DNA fragmentation: fragmentation of the DNA extracted from the sample in step (a) may be carried out by standard means known in the art. The sample DNA in this example was cleaved by NEBNext dsDNA Fragments from NEB, then recovered and purified by Gel Extraction Kit from OMEGA, and Fragments of about 100-250bp were collected. The concentration of the recovered fragmented DNA was 3.68 ng/. mu.L as determined by the Qubit 2.0 assay.

(c) Constructing a library: carrying out end repair and A tail addition on the fragmented DNA fragments; connecting a sequencing joint; and performing PCR amplification on the purified ligation product. In this example, Kapa Biosystems KAPA HTP library Perparation Kit was used for library construction. The method comprises the following specific steps:

(c-1) preparing a terminal repair reaction system as shown in Table 1, adding 20. mu.L of the terminal repair reaction system to each 1.5mL sample tube, mixing well, and incubating at 20 ℃ for 30 min.

TABLE 1 end-repair reaction System

(c-2) preparing 80% ethanol (40mL ethanol +10mL dH2O), wherein the 80% ethanol should be prepared as it is.

(c-3) after the end repair is completed, starting DNA purification, specifically comprising the following steps: firstly, adding 120 mu L of uniformly mixed magnetic beads into each 1.5mL sample tube, uniformly mixing a reaction system in a vortex manner, and placing at room temperature for 10min to fully combine DNA with the magnetic beads; then, placing a 1.5mL sample tube on a magnetic rack, and performing magnetic bead adsorption until the solution is clarified (generally waiting for 1-2 min); then, carefully removing the supernatant (20 uL of the supernatant can be remained at the bottom of the tube to avoid sucking magnetic beads), adding 500 uL of 80% ethanol, rotating the centrifuge tube at 180 ℃ to enable the magnetic beads to penetrate through the solution and be sucked to the tube wall of the other side, rotating for 2-3 times, or turning upside down and mixing uniformly for 6-8 times, standing for 15s, and then discarding the supernatant, wherein the centrifuge tube is kept on a magnetic rack all the time in the process; repeating the previous step once; and finally, taking down the centrifugal tube from the magnetic frame, quickly centrifuging, and then placing the centrifugal tube on the magnetic frame for separating again and removing the residual alcohol solution. And taking the centrifugal tube off the magnetic frame, opening the tube cover, drying the magnetic beads at normal temperature, and volatilizing ethanol to prevent excessive ethanol from influencing the effect of the enzyme in a subsequent reaction system. Here, the drying of the magnetic beads is carried out at room temperature, generally, the drying is carried out until the surfaces of the magnetic beads are not reflected or one or two seams are opened, and the process needs to wait for 2-3 min; the magnetic beads should be avoided from being too dry, which would result in a significant loss of DNA recovery.

(c-4) taking out the KAPA A-tailing buffer and the KAPA A-tailing enzyme reagent from a refrigerator at the temperature of 20 ℃ below zero, and placing the reagents on an ice box for thawing for later use. The metal bath was taken out of the 4 ℃ freezer and the temperature was adjusted to 30 ℃ for use. Then, a reaction system with a tail end added A was prepared as shown in Table 2. 50 μ L A-labeling master mix resuspended beads were added to each sample tube, mixed well and incubated at 30 ℃ for 30 min. After the end-addition reaction, DNA purification was started, and the same procedure as in (c-3) was carried out.

TABLE 2 Tail end A addition reaction System

(c-5) connecting both ends of the DNA fragment obtained in the step (c-4) with sequencing adapters, wherein the reaction system is shown in Table 3.

TABLE 3 linker attachment reaction System

First, 5 XKAPA Ligation buffer and KAPA T4DNA Ligation reagent were taken out from a freezer at-20 ℃ and placed on an ice box for thawing for use. The metal bath was placed in a refrigerator at 4 ℃ and the temperature was adjusted to 20 ℃ for future use. Then, 45. mu.L of linker-linked reaction system was added to each sample tube, and the beads were resuspended and mixed well. And then, according to the arrangement on the computer, the joint information corresponding to the sample record on the experimental record book. Taking out the temporary stored joint reagent at the temperature of 2-8 ℃. Here, a linker addition of less than 5. mu.L requires a volume of 5. mu.L to be filled with nucleic-Free water. Vortex well and mix evenly, react for 15min at 20 ℃. After the linker ligation reaction is completed, DNA purification is started, and the procedure is the same as in step (c-3).

(c-6) double sieving step: add 100. mu.L TE buffer to each sample tube, vortex and mix well, and let stand at room temperature for 5 min. mu.L of KAPA PEG/NaCl SPRI solution was added to each sample tube and allowed to stand at room temperature for 10 min. DNA fragments larger than 450bp were adsorbed onto magnetic beads. A new batch of 1.5mL centrifuge tubes is prepared, the tube walls of the tube caps are marked with corresponding numbers, and 20 mu L of uniformly mixed magnetic beads are added. The sample tube is placed on a magnetic rack for magnetic bead adsorption until the solution is clarified (generally waiting for 1-2 min). Carefully remove 155. mu.L of the supernatant, transfer it to a correspondingly numbered 1.5mL centrifuge tube containing magnetic beads, mix it by vortexing thoroughly, and let stand at room temperature for 10 min. DNA fragments larger than 250bp are adsorbed.

(c-7) after the double screening is finished, starting to purify the DNA, wherein the specific steps are the same as the DNA purification step. mu.L of nucleic-Free water was added to each sample tube, and the beads were resuspended, mixed well and allowed to stand at room temperature for 5 min. c-6-8: prepare a new batch of 0.2ml PCR tubes, and label the corresponding sample number on the tube cover. And (3) placing the sample tube in a magnetic frame, carrying out magnetic bead adsorption until the solution is clarified, and transferring the supernatant into the PCR tube with the corresponding number to be used as a template of the PCR experiment. And (3) preparing 199 mu L of the Qubitbuffer, adding 1 mu L of DNA sample, uniformly mixing by vortex, standing for 2min in a dark place, and measuring the concentration to be 0.19 ng/mu L.

(c-8) PCR amplification of the library: and performing PCR amplification by using the obtained complete double-stranded DNA adaptor connection product as a template. First, 30. mu.L of the PCR amplification reaction system shown in Table 4 was added to each 0.2mL sample tube, and vortexed to mix the mixture.

After the PCR reaction was completed, DNA purification was started, and the same operation as in step (c-3) was carried out. Then, the temperature and time parameters of the PCR amplification process were set as shown in Table 5. mu.L of nucleic-Free water (Nuclease-Free water) was added to each 1.5mL sample tube, mixed well and allowed to stand at room temperature for 5 min. A new batch of centrifuge tubes is prepared and the tube caps are labeled with information. And (3) placing a 1.5mL sample tube on a magnetic frame, carrying out magnetic bead adsorption until the solution is clarified, transferring the supernatant to a corresponding new 1.5mL centrifuge tube written with sample information, configuring a Qubit Buffer 199 mu L, adding a 1 mu L DNA sample, uniformly mixing by vortex, standing for 2min in a dark place, and measuring the concentration of 32.2 ng/mu L. The DNA library was tested for fragment size of 315bp using Agilent 2100. The library meets the quality qualification standard and enters a hybridization capturing link.

TABLE 4 PCR amplification reaction System

TABLE 5 PCR amplification Process temperature and time parameters

(d) And (3) hybridization and capture: and d, hybridizing the DNA library constructed in the step c with a probe, removing unbound DNA, and enriching target gene DNA fragments. It is noted that the capture probe for the DNA target fragment is a probe of the present invention. In this example, the hybridization Kit SeqCap EZ Library Kit from Roche was used, and the specific procedure was as described in the standard procedure of the specification.

(e) Library sequencing and data analysis:

(e-1) performing second-generation sequencing on the DNA library captured in the step (d), performing bioinformatics analysis on the sequencing result, and analyzing the variation condition of the target gene, including point mutation, gene fragment insertion/deletion, copy number change, fusion/gene rearrangement. In this example, the Illumina company nextsseq 500/550Kits v2 kit is used to complete sequencing on the Nextseq 500 sequencing platform, and the specific procedures refer to the standard procedures of the specification.

(e-2) the data analysis includes removing low quality data, clipping the data, and removing polyN error information. Removing low quality data includes removing mapping quality Q10 data; clipping the data comprises clipping and removing the last 8-10nt of reads; removing the polyN error information includes removing more than 20 consecutive polyN data in the data.

The final results are shown in tables 6 and 7, and the results show that the probe library prepared in the embodiment can simultaneously capture genes of single nucleotide polymorphic chains related to chemotherapeutic drugs, genes related to targeted drugs, genes related to stability of microsatellites for immunotherapy and viral genes, so that various disease course management schemes can be evaluated, and clinical treatment references can be provided for patients.

TABLE 6 partial genetic variation detected in HP201701 sample

TABLE 7 drugs associated with the genetic variation detected in HP201701 samples

While the invention has been described in detail in connection with the drawings and the embodiments, it is to be understood that the above description is not intended to limit the invention in any way. Those skilled in the art can make modifications and variations to the present invention as needed without departing from the true spirit and scope of the invention, and such modifications and variations are within the scope of the invention.

Sequence listing

<110> Shenzhen Shanpulos Biotech Co., Ltd

<120> method for detecting multiple tumor-associated genes for non-diagnostic therapeutic purposes

<160>19

<170>SIPOSequenceListing 1.0

<210>1

<211>115

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gtcacgtgac acagtcaaac attaacttgg tgtatcgatt ggtttttgcc atatatatat 60

atataagtag gagagggcga acctctggca ggagcaaagg cgccatggct gtgga 115

<210>2

<211>102

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

aattcagagt ctggggagga ggctcccaca ggccgggaca agaagcggaa gcagcagcag 60

cagcagcctg tgtagtctgc ccccgggaaa ctgaggaact aa 102

<210>3

<211>102

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tcttgcccag actcaacatg gcggctacac gtcgcctgtc agtctgtgaa gcctaccccg 60

ggcgtgggcc gcagcgtcga gtaacgtcat tcgaaccccg tc 102

<210>4

<211>157

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ggcggagcgc gggacggccg cgggaaaagg cgcgcggaag gggtcctgcc accgcgccac 60

ttggcctgcc tccgtcccgc cgcgccactt ggcctgcctc cgtcccgccg cgccacttcg 120

cctgcctccg tcccccgccc gccgcgccat gcctgtg 157

<210>5

<211>102

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tcagtccatg cctccacaga ggctatgcca gctgtaggcc agaccctggc aagatctggg 60

tggataatca gactgactgg tcccactctt cccacaggcc tc 102

<210>6

<211>137

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

caaattgtat ttacataatt acacactttg tctttgactt ctttttcttc tttttaccat 60

ctttgctcat cttttcttta tgttttcgaa tttctcgaac taatgtatag aaggcatcat 120

caacaccctg aaataca 137

<210>7

<211>108

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gggagcagcg atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc 60

gctctgcccg gcgagtcggg ctctggagga aaagaaaggt aagggcgt 108

<210>8

<211>120

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gctgacttac catgtgtctg ttttagaagt gaatattcct tccatgaagg cctctggggg 60

catccactta actggcagca tggcacagcc tccctttcta tagtagctcg ccctgtgggg 120

<210>9

<211>152

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

aaatatttac ctggtccctg ttgttgatgt ttgaataagg taactgtcca gtcatcaatt 60

catacagaac aattccaaat gcatatacat ctgactgaaa gctgtatgga tttttatctt 120

gcattctgat gacttctggt gcctgttaga ac 152

<210>10

<211>181

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gtccttgtag gttttcccaa atagtgcacc ccttgaagga gggacaaggc tgaccatatg 60

tggctgggac tttggatttc ggaggaataa taaatttgat ttaaagaaaa ctagagttct 120

ccttggaaat gagagctgca ccttgacttt aagtgagagc acgatgaata cgtaaggatc 180

t 181

<210>11

<211>265

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

atccacagct aacatcatac tcaataatga atgaccaaaa gcatttctct tatgataatg 60

gacaaaaaca ggatgcctgc ctttaacact gctattcaac attgctggaa gttctggcca 120

gagaaattag acacagtgat acacacacac acacacacac acacacacac acacacacac 180

acatatttta tagatagata gatggtatcc aagtcagaaa gggagaagta aaactatccc 240

tattgtagat gacacagtcc catag 265

<210>12

<211>214

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttgtgaaaat atttagtgcc actgaattat gcaataaaaa tggttaaaat gatatatttt 60

atattttgcc acaaaaatta ccaataacta gagacaggca gaaaaaacta cacacacaca 120

cacacacaca cacacacagt tgtgccagtg ccgatttcct gatttggatg tgctgtagtt 180

acatcagatg tgaccacggt gggaaactgg gtga 214

<210>13

<211>159

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gtgtaagcat ctgtgtatac tacgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 60

tgtgtgccac gtgtctccat ctggggtcgg catcagcacc tctgtgttac ttgcccagat 120

gagaatatgc atgccctgtg acactcttcc tagaccagg 159

<210>14

<211>275

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

acagaaatca tatttaccag gacacatctg ttaaataaaa gcattgtttc atgttggtgt 60

acgtctatac agggctatgt ataaccgact cctgtttctc ctccctgcaa ccacagaacc 120

atcacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 180

cacacacgga tacacgcaca gatacgctcc tttccacaaa tgcacgcaaa ccgggacgca 240

aacccacaac tcgagggctt agaccttcac tgctg 275

<210>15

<211>201

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ctctaaaaaa ggcaagcaga taaaagagaa cacgaaaaat attcctactc cgcattcaca 60

ctttctggtc actcgcgttt acaaacaaga aaagtgttgc taaaaaaaaa aaaaaaaaaa 120

aaggccaggg gagacataca tttaaatata aaaatagaac tgtgccagcg actccggctg 180

gaattctgct gaaagggatg t 201

<210>16

<211>176

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga ggaactactg 60

tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac 120

cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccg gaattg 176

<210>17

<211>173

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

atggctgatc ctgcaggtac caatggggaa gagggtacgg gatgtaatgg atggttttat 60

gtagaggctg tagtggaaaa aaaaacaggg gatgctatat cagatgacga gaacgaaaat 120

gacagtgata caggtgaaga tttggtagat tttatagtaa atgataatga tta 173

<210>18

<211>142

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

agaattcgtc ttgctctatt cacccttact tttcttcttg cccgttctct ttcttagtat 60

gaatccagta tgcctgcctg taattgttgc gccctacctc ttttggctgg cggctattgc 120

cgcctcgtgt ttcacggcct ca 142

<210>19

<211>147

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

ctccaccacg ttccaccaaa ctcttcaaga tcccagagtc agggctctgt actttcctgc 60

tggtggctcc agttcaggaa cagtgaaccc tgttcagaac actgcctctt ccatatcgtc 120

aatcttatcg aagactgggg accctgt 147

Claims (10)

1. A method for detecting a plurality of tumor-associated genes for non-diagnostic therapeutic purposes,

the method comprises the following steps:

(a) extracting DNA from the sample;

(b) constructing a DNA library based on the DNA of step (a);

(c) hybridizing the DNA library with a specially prepared probe library, purifying the hybridized library,

wherein the tailor-made probe library comprises a plurality of capture probes designed for a plurality of genes related to a plurality of tumor treatments, the 3 'ends of the plurality of capture probes are provided with biotin labels, and the biotin labels are configured at any position from 1 st to 5 th of the reciprocal of the 3' ends,

the multiple genes at least comprise a gene where a single nucleotide polymorphism chain related to a chemotherapeutic drug is positioned, a targeted drug related gene, a gene related to the stability of a microsatellite used for immunotherapy and a gene of a virus capable of causing cancer,

the gene related to the stability of the microsatellite for immunotherapy is used for judging a tumor-related label comprising tumor mutation load, the genes of the viruses comprise EBV virus related to nasopharyngeal carcinogenesis, HPV virus related to cervical cancer genesis, HBV and HCV virus related to liver cancer genesis,

the plurality of capture probes comprises: 1-19 of the sequence SEQ ID NO; probes designed for microsatellite stability-related genes for immunotherapy include SEQ ID NOs 11-15; probes designed for viral genes include SEQ ID NO 16-19; and is

(d) Sequencing the DNA result obtained in step (c) and detecting the genetic variation.

2. The detection method according to claim 1,

in the step (b), the DNA extracted from the step (a) is fragmented, and the fragmented DNA fragments are subjected to end repair and a-tailing, ligated to a sequencing adaptor and purified, thereby performing PCR amplification to obtain the DNA library.

3. The detection method according to claim 1,

the sample is a human sample.

4. The detection method according to claim 1,

in step (c), when the purification is carried out:

uniformly mixing the avidin-treated magnetic beads with the hybridized library to form a sample to be purified, and standing;

and (3) placing the sample to be purified on a magnetic rack for magnetic bead adsorption until the solution is clarified, washing the magnetic beads by using ethanol, and then drying the magnetic beads at room temperature.

5. The detection method according to claim 1,

the genetic variation includes at least one of point mutation, gene fragment insertion/deletion, copy number variation, and fusion/gene rearrangement.

6. The detection method according to claim 1,

in step (c), the length of the plurality of capture probes is 50-300bp, and the plurality of capture probes cover a target region and flanking sequences at two sides of the target region, wherein the target region is a DNA sequence of the plurality of genes, and the flanking sequences are sequences in a range of 5-50bp at two ends of the DNA sequence; and the number of the first and second electrodes,

the plurality of capture probes are designed in a shingled manner, and each of the plurality of capture probes has an overlap between them, the overlap being between 5 and 20 bases.

7. The detection method according to claim 1,

in step (d), performing data analysis on the result of the sequencing to detect genetic variation, the data analysis including removing unsatisfactory data from the result of the sequencing, the unsatisfactory data including data having a mapping quality (mapping quality) of less than or equal to Q10 and polyN data of 20 or more consecutive identical bases, and clipping the data result after the removal.

8. The detection method according to claim 2,

in step (b), the DNA extracted from step (a) is fragmented using a fragmenting enzyme or an ultrasonicator.

9. The detection method according to claim 2,

in the step (b), after the DNA fragments are connected with the sequencing adaptor, performing double screening by magnetic bead adsorption, wherein the double screening comprises the following steps:

adding a specified amount of buffer solution into a sample tube containing the DNA fragments, uniformly mixing by vortex, standing at room temperature, and adsorbing the DNA fragments by using magnetic beads until the solution is clear;

and taking out the supernatant from the sample tube, uniformly mixing the supernatant with the supernatant in a vortex manner, standing at room temperature, and adding the magnetic beads again to adsorb the DNA fragments.

10. The detection method according to claim 6,

in step (c), the length of the plurality of capture probes is 120bp, the flanking sequences are sequences within 10bp of both ends of the DNA sequence, and the overlapping part is 12 bases.

Images