CN113106149A - Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance - Google Patents
Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance Download PDFInfo
- Publication number
- CN113106149A CN113106149A CN202110453841.8A CN202110453841A CN113106149A CN 113106149 A CN113106149 A CN 113106149A CN 202110453841 A CN202110453841 A CN 202110453841A CN 113106149 A CN113106149 A CN 113106149A
- Authority
- CN
- China
- Prior art keywords
- fusion gene
- detection
- positive
- fusion
- fragments
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a fusion gene detection reference substance and application of the fusion gene detection reference substance. The method adopts negative DNA to dilute a fusion gene positive fragment library to obtain a fusion gene detection reference substance comprising at least 1 dilution gradient; the pool of fusion gene positive fragments comprises at least 11 fusion gene positive fragments. The invention adopts a plurality of fusion gene positive fragments, has wide detection coverage range, provides a new idea for overcoming the difficulty and more omission of DNA level fusion detection in the future, provides a detection sample plate for the performance evaluation standard of fusion gene detection products, provides a new index for the progress of fusion detection technology, and has wide application prospect and great industrial value.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a preparation method of a fusion gene detection reference substance and application of the fusion gene detection reference substance.
Background
Medically, cancer refers to a malignant tumor that originates in epithelial tissue, and is the most common type of malignant tumor. In 2020, lung, colorectal, breast and prostate cancer have been top ranked in terms of statistical data published by the american cancer society in the journal of clinical tumors. Due to the development of accurate medical treatment, gene detection is used as an effective means for tumor detection, and important information and technical support are provided for accurate treatment of tumors. It has been reported that the cause of solid tumor includes fusion of ALK, ROS1, RET, etc. genes, in addition to the already identified mutations of the driver genes, such as amplification of EGFR, KRAS, BRAF, etc. genes, and ERBB2, MET, etc. genes, resulting in the formation of fusion genes.
The term "fusion gene" refers to a gene-level fusion of a certain protooncogene and another gene, i.e., a partner gene. The fusion gene can drive the synthesis of proteins which promote cell proliferation in cells in a large amount, so that the cells are proliferated in a large amount, and finally, the cancer is generated and developed. The gene fusion is an important genetic change of hematological malignancy and solid tumor, for example, the BCR-ABL fusion gene is an important index which needs to be detected during diagnosis/treatment of chronic myelogenous leukemia; the EML4-ALK positive non-small cell lung cancer patient is also used as the basis for the crizotinib.
In non-small cell lung cancer, common fusion genes comprise ALK (3% -7%), ROS1 (1% -2%), NTRK (3.3%) and RET (0.7% -2%), and the genes are closely related to the occurrence and development of cancer and targeted drug therapy. ALK fusion is one of the key drivers of non-small cell lung cancer, occurring at a frequency of about 3-7% in NSCLC patients. The inhibitors of crizotinib, ceritinib and Alectinib aiming at ALK fusion have great success in treating NSCLC patients positive to ALK fusion, and ALK fusion detection is a precondition for the ALK treatment. The rearrangement of the ROS1 gene was initially identified in the human glioma cell line, and the ROS1 gene rearrangement was subsequently found in several other malignancies, such as cholangiocarcinoma, ovarian cancer, gastric cancer and non-small cell lung cancer, wherein the mutation frequency in non-small cell lung cancer is 1% -2%. Rearrangement of ROS1 results in sustained kinase activation, up-regulation of SHP-1, SHP2, and PI3K, AKT, mTOR, MAPK, and ERK signaling pathways, leading to sustained cell proliferation and tumorigenesis. The kinase active regions of ALK and ROS1 share 70% similarity, so many ALK inhibitors are available for the treatment of ROS1, and there is currently approval for the targeted drug crizotinib. Another common fusion gene is the RET gene, the RET rearrangement first found in 2012 in NSCLC, and at least 12 fusion variants have been found to date, of which KIF5B-RET is the most common type. The KIF5B-RET fusion gene is a novel oncogenic driving mutation and exists in a part of non-small cell lung cancers, the ORR 16% -47% of RET fusion NSCLC is treated by the existing multi-target kinase inhibitor, and the PFS lasts for 2.3-7.3 months. In addition to the NTRK gene, the normal NTRK gene is responsible for encoding Tropomyosin Receptor Kinase (TRK) protein and functions to maintain the survival and normal function of nerve cells. However, if fusion mutation occurs in NTRK, abnormal TRK protein is encoded, and cells are abnormally proliferated, thereby causing tumor. NTRK fusions including NTRK1, NTRK2, or NTRK3 gene fusions have been found to be involved in the development of a variety of tumors. NTRK fusion has been reported in almost every cancer, although the mutation rate is less than 5% in common tumors such as lung cancer, colorectal cancer. Especially in some rare tumors, such as salivary gland cancer, infantile fibrosarcoma, mutation rate is over 90%. Larotrectinib is the only targeted drug approved for NTRK fusion at present, and is targeted to tumor patients with NTRK1, NTRK2 or NTRK3 gene fusion. The EGFR gene is mainly used for quantitative detection of a fusion fragment library as a non-fusion gene.
At present, the following detection techniques are mainly used for detecting fusion genes: (1) fluorescence in situ hybridization detection (FISH), a gold standard for gene fusion detection. The fluorescence in situ hybridization can detect the location of a target gene on a DNA layer in a chromosome so as to judge whether the occurrence of gene ectopy exists, but the experimental operation is complex, the technical requirement is higher, the cells can be only visually observed under a microscope, the observed cells are limited, and the condition of missing detection can exist. (2) The immunohistochemical detection technology (IHC) is one of the common methods for clinical detection at present, and is a method for detecting the expression level of a target fusion protein by combining a specific antibody with the fusion protein, which can detect whether gene fusion occurs or not from a protein level and can quantify the expression of the fusion protein, but has the defects that the fusion gene cannot be directly detected from a DNA level usually, and the immunohistochemical method has strong subjectivity on the result, so that high false positive rate is easily caused. (3) Reverse transcription qPCR (RT-qPCR) detection has the advantages of strong operability, simple and rapid detection and judgment by designing a PCR primer aiming at a known fusion gene, obvious defects, high requirement on the quality of an RNA sample and easy influence on the result of the RNA sample due to the fact that RT-qPCR only aims at the detection of the RNA sample.
The three techniques are only suitable for detecting tumor tissue samples, and limit the application expansion, because many patients with advanced cancer cannot be subjected to surgical sampling, and therefore the detection techniques cannot be used for detecting the state of the fusion gene.
Since the high throughput sequencing (NGS) detection technology can detect multiple genes and multiple fusion variants at one time and discover unknown variation, the corresponding detection panel has been approved by NMPA at present and can be used for clinical detection. Although the detection of fusion genes by high-throughput sequencing has great technical advantages and development prospects, many problems still exist from the viewpoint of DNA-level detection. One is that the detection performance of common fusion gene detection products on the market is uneven, and the condition of missing detection is easy to occur in the detection of puncture tissues or pathological samples with low tumor cell content. And secondly, the detection performance of the fusion gene detection product is not evaluated by a unified standard or standard in the industry, so that a reference substance or a standard substance is required to be provided for detecting the fusion gene detection product and is used as a basis for evaluating the fusion gene detection product.
Various fusion reference products are available on the market at present, but the following problems still exist: one is that the existing fusion reference products have few detection sites, and the existing known fusion gene detection reference products can only detect the fusion mutation of a certain specific site of one gene or two genes by each reference product, so that the actual performance of the fusion gene detection product cannot be objectively evaluated, and the detection efficiency is low. Secondly, the preparation process of the multi-site fusion reference substance is complex, and the conventional preparation process of the fusion reference substance comprises the steps of embedding a mutant cell line, performing transfection to obtain genome extraction of the mutant monoclonal cell line, and recombining clone plasmids. However, these existing preparation processes are only suitable for preparing less than 10 sites, and the process for preparing a multi-site fusion reference substance with more than 10 sites is complicated. Thirdly, the quality control cost of the DNA multi-site fusion reference product is high, and the quality control difficulty is high. The current fusion reference substance is prepared and then is accurately quantified at each site of each frequency by adopting a ddPCR mode, so that the quantification accuracy of the reference substance is determined. However, for the fusion reference products with multiple sites, due to the particularity of the ddPCR quantification mode, each site needs to be quantified separately, and the quantification efficiency is low; moreover, each fusion gene needs a specific ddPCR probe and primer, so that the actual operation cost is high and the difficulty is high, and in addition, partial DNA fusion genes often occur in a repetitive region sequence, so that the ddPCR quantitative result has unreliability. Therefore, there is no simple and efficient means for quantifying the amount of the multi-site fusion reference. And fourthly, the evaluation of the gene detection panel by the existing DNA fusion reference products on the market is in doubt. The size of a common fusion occurrence region of a common fusion gene of a solid tumor, such as ALK, ROS1, RET and the like, is 4000-10000bp, and the existing DNA fusion reference product can evaluate the fusion detection performance of the whole gene only by evaluating the fusion detection of one to two specific sites in the period, so that the authenticity of an evaluation result is questioned in a partial and comprehensive evaluation mode. Fifthly, the test cost of the fusion gene detection product is high, and because of the existing known fusion gene detection reference products, the number of detection sites of each reference product is only 1 to 10. Aiming at the test of large panel or multi-fusion gene detection products, a large number of reference products of different genes and different fusion mutation sites are needed for testing, so that the workload of research and development personnel for product performance detection is greatly increased, and the test cost of the fusion gene detection products is increased. And sixthly, various detection products, namely the fish dragon, in the NGS fusion detection market are mixed, and an effective reference product is lacked for evaluating the fish dragon.
In order to better evaluate the detection performance of high-throughput sequencing on fusion genes and optimize a DNA fusion gene detection product or a detection method system, a reference product which is convenient and rapid in preparation process, simple and efficient in quantitative method, capable of covering a plurality of sites and capable of more accurately evaluating detection of fusion genes such as ALK, ROS1, RET, NTRK and the like related to solid tumors is needed.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a high-throughput sequencing method or application of ddPCR (double-stranded polymerase chain reaction) in preparation or detection of a fusion gene positive fragment library or a detection fusion gene detection reference product, provides a preparation method of the corresponding detection fusion gene positive fragment library and the fusion gene detection reference product, provides the fusion gene positive fragment library and the fusion gene detection reference product obtained by the method, further provides application of the obtained fusion gene detection reference product, provides a new thought for overcoming difficult and missed detection of DNA (deoxyribonucleic acid) level fusion detection in the future, and provides a detection sample plate for the standard of the fusion gene detection product.
In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:
in a first aspect, the invention provides the use of a high throughput sequencing method in the preparation or detection of a pool of fusion gene positive fragments, or in the preparation or detection of a fusion gene detection reference.
In combination with the first aspect, the invention provides a preparation or detection method of a fusion gene positive fragment library, wherein the preparation or detection method comprises the step of detecting the quantity ratio of each positive fragment in the fusion gene positive fragment library obtained by mixing different positive fragments by using a high-throughput sequencing method. The preparation method of the fusion gene positive fragment library also comprises the step of adjusting the number of each positive fragment in the fusion gene positive fragment library.
In an alternative embodiment, the detecting step by the high-throughput sequencing method comprises performing disruption, repair, ligation and purification on each positive fragment in the fusion gene positive fragment library in sequence, and then performing homogeneity evaluation on each positive fragment by the high-throughput sequencing method.
Preferably, the homogeneity evaluation comprises evaluating the copy number of each positive fragment in the pool of fusion gene positive fragments.
Preferably, the copy number of each positive fragment is obtained by detecting the sequence depth.
In the process of evaluating the homogeneity of the fusion gene positive fragments, the sequencing depth of the fusion gene positive fragments obtained by high-throughput sequencing is used as a standard for evaluating the copy number of the fusion gene positive fragments, and the content of different fusion gene positive fragments can be estimated through the evaluation of the copy number, so that basis and reference are provided for the quantitative determination of the use amount of the fusion gene positive fragments by adopting ddPCR in a subsequent reference substance preparation method, the mutation frequency of each fusion gene positive fragment after the fusion gene detection reference substance is prepared can be accurately calculated, and compared with the direct ddPCR detection quantification, the detection efficiency can be greatly improved.
In a second aspect, the present invention also provides a fusion gene positive fragment library obtained by the preparation method described in the previous embodiment.
Preferably, each positive fragment in the pool of fusion gene positive fragments is mixed equimolar.
Preferably, the total concentration of the fusion gene positive fragments in the fusion gene positive fragment library is 2-10 ng/mu L.
In a third aspect, the invention also provides application of ddPCR in preparation or detection of a fusion gene detection reference.
In combination with the third aspect, the invention provides a preparation or detection method of a fusion gene detection reference, the preparation or detection method comprises the steps of detecting the mutation frequency of each positive fragment in the fusion gene detection reference by ddPCR, and the fusion gene detection reference comprises a fusion gene detection reference obtained by diluting a mixture of a marker locus positive fragment and a fusion gene positive fragment by negative DNA; the preparation or detection method further comprises the step of detecting the quantity ratio of the positive fragments in the fusion gene detection reference substance by adopting a high-throughput sequencing method.
Preferably, the number of marker site positive fragments is at least 2.
In a fourth aspect, the present invention also provides a fusion gene detection reference substance obtained by the foregoing embodiment.
Preferably, the fusion gene detection reference comprises a tumor tissue reference, and the source of negative DNA of the tumor tissue reference comprises a wild-type GM12878 cell line.
Preferably, the negative DNA of the tumor tissue reference is derived from gDNA of a wild-type GM12878 cell line;
preferably, the tumor tissue reference comprises at least 3 dilution gradients.
Preferably, the tumor tissue reference dilution gradient comprises 5%, 2% and 1% fusion gene positive fragments as a percentage of total DNA moles.
The tissue reference substance containing 5%, 2% and 1% gradient fusion mutations can be used as a detection limit reference substance for multi-site fusion gene detection, can also be used for testing different detection limit requirements of detection panel with different sizes at different sequencing depths, and the preparation process flow for constructing the fusion gene detection reference substance by doping gDNA of a GM12878 cell line into a fusion gene positive segment with a certain proportion is simple, the GM12878 cell line is derived from human B lymphocytes, and the human source of the obtained fusion gene detection reference substance is good.
Preferably, the fusion gene detection reference comprises a plasma reference obtained by ultrasonically cutting the fusion gene positive fragment, and the negative DNA of the plasma reference comprises plasma negative DNA.
Preferably, the dilution gradient of the plasma reference substance comprises the total moles of fusion gene positive fragments accounting for 1%, 0.5% and 0.2% of the total moles of DNA, and the plasma cell DNA fusion gene detection reference substance containing 1%, 0.5% and 0.2% of three gradient fusion mutations can be suitable for testing different detection limit requirements.
In alternative embodiments, the marker site positive fragments selected for use in the previous embodiments include SNP positive fragments.
Preferably, the number of the SNP positive fragments is 2, as shown in Table 1.
The positional information of SNP positive fragments shown in the positive fragments 81 and 82 in Table 1 are as follows:
the SNP positive fragment name "COSM 6241-SNP _ 1" in Table 1 indicates that the base mutation in the positive fragment 81 is numbered 6241 in the cosmic database, and the SNP positive fragment name "COSM 6224-SNP _ 2" indicates that the base mutation in the positive fragment 82 is numbered 6224 in the cosmic database.
The positional information expression of the positive fragment 81 in Table 1 is "chr 7:55248593-55249592(+) _55249005G > T (S768I)", which indicates that the SNP positive fragment is a forward sequence of a sequence region having a coordinate position of 55248593 to 55249592 on chromosome 7 of the corresponding gene, and the base G having a coordinate position of 55249005 is mutated to T, which mutation results in the mutation of the 768 th amino acid of the corresponding amino acid sequence from S to I.
The positional information expression of the positive fragment 82 in Table 1 is "chr 7:55258975-55259974(+) _55259515T > G (L858R)", which indicates that the SNP positive fragment is a forward sequence of a sequence region with the coordinate positions of 55258975 to 55259974 on the chromosome 7 of the corresponding gene, and the base T with the coordinate position of 55259515 is mutated to G, and the mutation results in the mutation that the 858 th amino acid of the corresponding amino acid sequence is mutated from L to R, and the base T at the base position of 2585 of the coding region is mutated to A.
In an alternative embodiment, the fusion gene positive fragment library or the fusion gene detection reference product according to the previous embodiment of the present invention comprises at least 11 fusion gene positive fragments from positive fragments 1 to 80 shown in table 2, and the interval between the corresponding gene loci of the different fusion gene positive fragments is greater than 300 bps.
TABLE 2 fusion gene positive fragment information provided by the present invention
The genome version of the source gene reference is Feb.2009(GRCh37/hg 19).
The general formula of the name of the fusion gene positive fragment in table 2 is "GeneA- > GeneB _ x", which indicates that gene a and gene B are fused, and gene a is located on the left side of the breakpoint, gene B is located on the right side of the breakpoint, and "x" indicates the number of the fusion gene positive fragment in the fusion gene positive fragment library.
The expression of the position information of the fusion gene positive fragment in table 2 is "chromosome number a: site a-site B (+/-) _ chromosome number B site c-site d (+/-) ", which indicates that the sequence interval from site a to site B of chromosome A of gene A is linked to the sequence interval from site c to site d of chromosome B of gene B," (+/-) "indicates that the sequence interval is in positive order (+) or negative order (-).
For example, the fusion gene name of positive fragment 1 is: EML4- > ALK _1, which indicates that EML4 and ALK are fused, the left side of a breakpoint of the fusion gene is an EML4 gene, the right side of the breakpoint is an ALK gene, the number of the fusion positive fragment in a fragment library is 1, the information of the corresponding fusion gene positive fragment is chr2: 42543176-.
In addition, a base G and a base C are connected between two gene segments connected in the fusion gene positive segment information of the positive segment 5 and the positive segment 73 in the table 2, respectively, which indicates that the base G and the base C are inserted at the break points of the fusion gene positive segments 5 and 73, respectively.
The fusion gene positive fragment 1-80 is a gene sequence derived from ALK, ROS1, RET, NTRK2 and NTRK 3.
The fusion gene positive fragment is obtained by artificial synthesis, and the DNA fusion positive fragment is constructed by using an in-vitro artificial DNA synthesis mode, so that the method is high in speed and high in flux compared with a conventional PCR amplification combined carrier construction mode.
In a fourth aspect, the present invention also provides a use of the fusion gene detection reference substance provided in the preceding embodiment in any one of (a) to (E):
(A) as a reference for DNA fusion gene detection;
(B) evaluating a DNA fusion gene detection product based on a high-throughput sequencing platform;
(C) calibrating the detection result of the DNA fusion gene;
(D) optimizing a DNA fusion gene detection product or a detection method system;
(E) the stability of tumor panel was verified and optimized.
The invention has the following beneficial effects:
the fusion gene positive fragment library provided by the invention comprises at least 11 different fusion gene positive fragments from 1-80 positive fragments, and the 80 fusion fragment sequences cover a plurality of exon regions of ALK, ROS1, RET, NTRK2 and NTRK3 genes. Compared with the prior art that the fusion reference substance generally selects one or more single sites of the gene, the fusion gene positive fragment library provided by the invention can cover a plurality of sites of a plurality of genes, has a wide detection range, can better evaluate the detection performance of a DNA fusion detection product, and ensures the accuracy of the detection result because the interval between the corresponding gene sites of different fusion gene positive fragments is more than 300 bp.
The fusion gene detection reference product prepared by mixing the fusion gene positive fragment library and the negative DNA can be used as a reference product for DNA fusion gene detection, evaluating a DNA fusion gene product based on a high-throughput sequencing platform, calibrating a DNA fusion gene detection result, optimizing the DNA fusion gene detection product or a detection method system and the like, provides a new idea for overcoming the defects of difficult and many missed detections of DNA level fusion detection, provides a detection sample plate for the standard of the fusion gene detection product, provides a new index for the progress of the fusion detection technology, and has wide application prospect and huge industrial value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the preparation process of an NGS PCR-Free library, a tissue reference and a plasma reference in an alternative embodiment of the present invention;
FIG. 2 is a graph showing the detection results of ddPCR in example 5;
FIG. 3 is a diagram showing the quality control test results of Agilent 4200 in example 6.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In an optional embodiment, the preparation process of the NGS PCR-Free library (fusion gene positive fragment library), the tissue reference product and the plasma reference product is shown in fig. 1, positive fragments are designed and synthesized according to sequences shown in positive fragments 1-80, the obtained positive fragments are prepared into positive fragment stock solutions, concentration quantification is performed on 80 parts of the stock solutions, then 80 parts of the positive fragments are mixed in an equimolar mode, SNP positive fragments of the sequences shown in the positive fragments 81 and 82 are added, and the NGS PCR-Free library is obtained by quantifying the positive fragments by NGS sequencing with the SNP positive fragments as references.
And after extracting the equimolar mixed positive fragments from the negative DNA of a wild GM12878 cell line, mixing and diluting with expected VAFs and concentration, quantifying by ddPCR to obtain a tissue reference substance, and performing NGS sequencing and data analysis on the obtained tissue reference substance to obtain a qualified tissue reference substance.
And ultrasonically cutting the tissue reference product subjected to ddPCR quantification to obtain a plasma reference product capable of being used for plasma sample detection, and performing NGS sequencing and data analysis on the obtained plasma reference product to obtain a qualified plasma reference product.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Design of fusion Gene Positive fragments
(1) Common solid tumor target drug fusion genes are selected as development targets, and the target genes are determined as follows: ALK, ROS1, RET, and NTRK, and the SNP site of EGFR was additionally selected as the site for ddPCR quantification.
(2) And logging in a COSMIC database, and inquiring related sequence information of all fusion breakpoints of ALK, ROS1, RET and NTRK genes and sequence information of EGFR SNP sites.
(3) Designing fusion gene positive fragments according to the information of the DNA breakpoints of the fusion gene: the chaperone gene 500bp + fusion gene 500bp is adopted for design, and the total length of the fusion gene positive fragment is 1000 bp. Designing SNP gene positive fragments by using the information of the SNP sites at the EGFR SNP site: the two sides of the SNP locus are respectively designed in a 500bp mode, and the total length is 1000 bp.
(4) Optimization of fusion gene positive fragment design: in order to avoid mutual interference among various fusion gene positive fragments, all the fusion gene positive fragments with the breakpoint interval of more than 300 bases are screened out as finally determined sequences.
(5) Finally, 80 fusion gene positive fragments are obtained, as shown in Table 2, and 2 SNP positive fragments are obtained, as shown in Table 1.
The embodiment provides 82 positive fragments in total, the detection coverage is wide, the detection coverage comprises 6 ALK genes, 20 ROS1 genes, 4 RET genes, 13 NTRK2 genes, 37 NTRK3 genes and 2 EGFR genes, and COSM6241-SNP _1 and COSM6224-SNP _2 can be used for sites of ddPCR detection, and the fusion gene positive fragment library can be accurately quantified, so that the detection result obtained when the fusion gene detection reference prepared by the fusion gene positive fragment library provided by the embodiment is used for fusion gene detection is more accurate.
Example 2
This example provides a method for constructing a pool of fusion gene positive fragments using the fusion gene positive fragments provided in example 1, comprising the steps of:
2.1 Synthesis of fusion Gene Positive fragment: 82 fusion gene positive fragments designed in example 1 were submitted to Twist Bioscience for double-stranded DNA fragment synthesis.
2.2 preparation of a stock solution of fusion gene positive fragments: after receiving dry powders of fusion gene positive fragments synthesized by Twist Bioscience, each of the dry powders of positive fragments was dissolved in EB buffer solution, and prepared into stock solutions.
2.3 determination of concentration of fusion gene positive fragment: measuring the DNA concentration of each positive fragment by using a Qubit4.0 nucleic acid quantifier, and calibrating the instrument by using a standard substance before measuring the concentration; for concentration determination, duplicate detection was performed for each positive fragment.
2.4 preparation of fusion gene positive fragment library:
(1) according to the determined concentration of each positive fragment, 6.32X 10 of each positive fragment is calculated-14The volume required for moles.
(2) And selecting a low-adsorption gun head and an EP (ethylene propylene) tube for pipetting operation.
(3) And (3) determining the total concentration of the fusion gene positive fragments in the mixed fusion gene positive fragment library, wherein the detection concentration is as follows: 4.6 ng/. mu.L, and labeling to complete the preparation of the fusion gene positive fragment library.
Example 3
This example provides a method for quantifying copy number of fusion gene positive fragments in the fusion gene positive fragment library obtained in example 2, comprising the following steps:
3.1 library construction of fusion Gene Positive fragment library
(1) Breaking and repairing tail ends: the fusion gene positive fragments in the fusion gene positive fragment library obtained in example 2 were disrupted and end-repaired on a PCR instrument.
(2) And (3) connection reaction: and (3) performing joint connection on the repaired DNA.
(3) Magnetic beads are used for purification, 1 mu L of purified product is taken, a nucleic acid quantitative analyzer of Qubit4.0 is used for measuring the DNA concentration, and the detection concentration range is as follows: 4.6 ng/. mu.L, completing the construction of the NGS PCR-Free library.
3.2 high throughput sequencing was performed on the NGS PCR-Free library and the sequencing results were used for sequence analysis.
3.3 fusion Gene Positive fragment library sequence analysis
(1) Design of reference Gene sequences
According to the base sequence information in the fusion gene positive fragment library provided in the gene positive fragment information table 1 and table 2 provided in example 1, 150 bases at both ends of the fusion breakpoint are extracted as the reference gene sequences to be aligned.
(2) And (3) data quality control: and performing quality control on the high-throughput sequencing data by using Fastp, filtering a sequencing joint, a low-quality base, a sequencing error fragment and the like, and obtaining high-quality data (clean data) after filtering.
(3) Alignment of sequences: completely comparing clear data with a reference gene sequence by using comparison software bwa, and counting the number of reads of each fusion gene positive fragment; then, the gencore software is used for comparing and processing the data by de-duplication and base correction.
(4) Calculation of homogeneity: depth statistics of the reference sequence is performed by using the bedtools coverage, and an average value of actual detection values of each group is calculated and named as: average sequencing depth; calculating the standard deviation of each group of actual detection values; and dividing the obtained standard deviation by the average sequencing depth to obtain the CV value of each group of actual detection value data.
(5) Detection and sequencing analysis: the NGS PCR-Free library of the fusion gene positive fragment library obtained in the step 3.1 is used for confirming the uniformity of the positive fragment doped in the NGS PCR-Free library through high-throughput sequencing detection, and the results are shown in a table 3.
TABLE 3 results of detection of the number of reads of 82 positive fragments in example 3
The statistical result shows that the CV value of each group of actual detection values is between 10% and 15%, which indicates that all positive fragments in each group have good uniformity and the results of three experiments are stable. Therefore, the fusion gene positive fragment library to-be-detected sample prepared by the experiment can be used as the fusion gene positive fragment library for preparing subsequent reference substances.
In addition, in the embodiment, the quantification of the positive fragments is performed by adopting the NGS sequencing, and compared with a mode of quantifying each site one by the traditional ddPCR, the NGS sequencing quantification is a simple and efficient means, i.e., the fusion reference substance of multiple sites can be quantified quickly without considering the sequence complexity of the fusion gene and a large amount of primer probe design work.
Example 4
This example provides a method for preparing a tumor tissue fusion gene reference (hereinafter referred to as a tissue reference), wherein the tissue reference is obtained by diluting the fusion gene positive fragment library provided in example 2 with gDNA of GM12878 cell line, and specifically includes the following steps:
4.1 extracting gDNA of the GM12878 cell line cultured in vitro by using the kit, then measuring the DNA concentration of the extracted gDNA of the cell line by using a nucleic acid quantifier of Qubit4.0, and calibrating the instrument by using a standard substance before measuring the concentration; when the concentration is measured, repeated detection is performed once.
4.2 negative cell gDNA was mixed with the pool of fusion gene positive fragments provided in example 2 to obtain 5 dilution gradients of tissue reference. Wherein 5, 2, 1, 0.5 and 0.2 mole percent of the tissue reference was used for the preparation of the present example and 1, 0.5, 0.2 mole percent of the tissue reference was used for the preparation of the plasma reference of example 6.
Example 5
This example provides a method for performing frequency quantification of the tissue reference prepared in example 4, comprising the steps of:
5.1 the tissue reference obtained in example 4 was subjected to detection of mutation frequency by ddPCR using ddPCR detection technique of BIO-RAD QX200 platform, and the detection sites are shown in Table 4:
TABLE 4 detection sites for ddPCR in example 5
| Gene locus | Type of mutation | COSMIC numbering |
| EGFR L858R | SNP | COSM6224 |
| EGFR S768I | SNP | COSM6241 |
| CD74->ROS1 | Fusion | COSM1201 |
The primers and probes used in the ddPCR detection procedure are shown in Table 5.
TABLE 5 primers and probes used for ddPCR detection in example 5
The ddPCR reaction system composition is shown in Table 6:
TABLE 6 Table of the composition of ddPCR reaction system in example 5
| Components | Stock Con. | Final Con. | 1xVolume(uL) | |
| ddPCR supermix | | 1x | 10 | |
| Primer mix | 40x | 1x | 0.5 | |
| Probe mix | 40x | 1x | 0.5 | |
| Tumor tissue fusion reference substance | 15-50ng | - | X | |
| Buffer EB | - | - | 9-X | |
| Final volume of reaction | 20 |
5.2 droplet generation: adding the mixed reaction system into a middle row hole of a microdroplet generation card, adding microdroplet generation oil into a lower row hole, wearing a rubber leather sleeve, placing the microdroplet generation card into a microdroplet generation instrument, and waiting for microdroplet generation.
5.3 droplet transfer: the microdroplets generated in step 5.2 were transferred to a 96-well PCR plate and information was recorded for each sample applied to each well.
5.4 sealing the membrane: the 96-well PCR plate was sealed with a heat seal film machine.
5.5PCR reaction: the 96-well plate was placed on the PCR instrument, the lid was closed, the instrument ramp rate was adjusted to 2.5 ℃/s, and the program was run as per table 7:
TABLE 7 DDPCR run procedure in example 5
5.6 Signal Collection: after the PCR instrument run was completed, the 96-well plate was transferred to the QX200 droplet reader, the software "quantatasoft" was opened, the sample reader was edited, and data reading was started.
5.7 data analysis: and when the data reading is finished, opening the generated data reading file, and selecting 'Analyze' to Analyze the result.
The results of the tests are shown in fig. 2 and table 7, and the test frequency was in agreement with the expectation when 3 replicates were performed for each concentration of the reference. Therefore, the mixed DNA sample after ddPCR detection can be used as the tissue reference substance of the invention.
TABLE 7 tissue reference ddPCR assay results in example 5
Example 6
The embodiment provides a preparation method of a plasma fusion gene detection reference product (hereinafter referred to as a plasma reference product), wherein the plasma reference product is obtained by performing ultrasonic interruption on the tissue reference product provided in embodiment 4, and the preparation method specifically comprises the following steps.
6.1, subpackaging and diluting: 10ug of 1%, 0.5% and 0.2% tissue reference samples qualified by detection were taken out, respectively, and the concentrations thereof were diluted to 50 ng/. mu.L.
6.2 ultrasonic breaking: subpackaging the diluent obtained in the step 6.1 into each ultrasonic breaking tube according to 50 mu L/tube, carrying out ultrasonic breaking according to an ultrasonic breaking program shown in a table 8, and obtaining breaking products after breaking.
Table 8 ultrasonic instrument operating procedure in example 6
Remarking: the ultrasonic treatment is carried out for 7 cycles, then for 7 cycles, and for 21 cycles.
6.3, purification: and adding 1.5X Agencour AMPure XP magnetic beads into the interruption product, purifying the interruption product, and obtaining a plasma reference substance sample to be detected after purification.
6.4 detection and analysis of plasma reference: and (3) carrying out quality control detection on the size of the plasma reference substance fragment of the plasma reference substance sample to be detected in the step 6.3 by an Agilent 4200 bioanalyzer.
The detection result is shown in FIG. 3, and FIG. 3 shows that the prepared plasma DNA fusion detection reference substance is detected by Agilent 4200 bioanalyzer to obtain an electrophoretogram, and the obtained DNA fragment has a size of 150-220bp, which is closer to the size of the fragment of the real plasma DNA. Therefore, after the quality control of the Agilent 4200 bioanalyzer is confirmed, a qualified plasma fusion gene detection reference product is obtained.
Example 7
This example provides a method for preparing an NGS pre-library of tissue references obtained in example 5, which specifically includes the following steps.
7.1 disruption and end repair: tissue references were interrupted and end-repaired on a PCR instrument.
7.2 ligation: and connecting the repaired tissue reference products by joints.
7.3 Pre-library amplification: and carrying out PCR amplification on the tissue reference product connected by the joint to obtain enough DNA fragments with joints, namely the pre-library.
7.4 Pre-library purification and concentration determination: purification was performed using purified magnetic beads. Mu.l of the pre-library purified product was taken and concentration determined using a Qubit4.0 nucleic acid quantifier.
Example 8
The present example provides a method for preparing an NGS pre-library of a plasma fusion reference, which is obtained in example 6 and specifically includes the following steps.
8.1 end repair: samples were end-repaired on a PCR instrument.
8.2 ligation: and (3) performing joint connection on the repaired DNA.
8.3 Pre-library amplification: and carrying out PCR amplification on the product after the joint connection to obtain enough DNA fragments with joints, namely the pre-library.
8.4 Pre-library purification and concentration determination: purification was performed using purified magnetic beads. Mu.l of the pre-library purified product was taken and concentration determined using a Qubit4.0 nucleic acid quantifier.
Example 9
This example provides a method for hybrid capture of the NGS pre-libraries obtained in examples 7 and 8 by fusion gene detection panel and final library acquisition, which specifically comprises the following steps.
9.1 hybridization reaction: the probe-bound sample was captured using streptavidin magnetic beads.
9.2 Final library amplification: and carrying out PCR amplification on the DNA fragments captured by the magnetic beads to obtain enough labeled DNA fragments, namely the final library.
9.3 library quality control: and (3) carrying out magnetic bead purification on the final library, carrying out concentration measurement and fragment quality inspection, and carrying out quantification by utilizing qPCR.
9.4 sequencing.
Example 10
This example provides a method for performing NGS sequencing and assay for final plasma/tissue fusion reference library obtained in example 9, which specifically includes the following steps.
(1) And performing quality control on the obtained second-generation sequencing data by using quality control software fastp, filtering sequencing joints, low-quality bases, sequencing error fragments and the like, and filtering to obtain high-quality data (clean data).
(2) Comparing clean data with a reference genome hg19 by using comparison software bwa to obtain corresponding specific position information on the genome of each DNA fragment; then, the gencore software is used for comparing and processing the data by de-duplication and base correction.
(3) Structural variant events were reported using pasv (pull all sv). The paSV inputs the name of the parameter sample, the bam path after the comparison of the steps, the used PANEL, the type of the sample (DNA/RNA), the output path and the catalog of the dependent program in turn.
(4) And (4) reporting the SVscan _ report.xls file of the result in a classified manner. And counting and outputting supported reads in sr _ all.
The high-throughput sequencing fusion detection results are shown in the table below, and related fusions can be effectively detected when detecting 1%, 2%, 5% tissue reference substances and 0.2%, 0.5%, 1% plasma reference substances.
The detection results of tumor tissue fusion gene reference products with the molar percentages of 1%, 2% and 5% are shown in table 9:
TABLE 9 detection results of tumor tissue fusion gene reference
The results of the detection of the plasma fusion gene reference products with the mole percentages of 0.2%, 0.5% and 1% are shown in table 9:
TABLE 9 plasma fusion Gene reference assay results
Comparative example 1
The fusion reference of this comparative example was subjected to high throughput sequencing using the fusion assay product of example 9 using a commercial 5% tissue DNA fusion reference.
The fusion assay results are shown in the following table:
TABLE 10 commercial fusion reference assay results
In the comparative example, the fusion breakpoint of the EML4-ALK gene is: chr2:29446974, which is more consistent with the breakpoint of the example positive fragment ALK-EML4_ 3; in addition, the breakpoint chr6:117646729 of CD74- > ROS1 in the comparative example is consistent with the example positive fragment ROS1-CD74-ddPCR test-71. ALK and ROS1 gene-related data were extracted from example 9 and comparative example 10, respectively, and compared.
TABLE 11 comparison of the test results of the examples and comparative examples
According to the comparison result, the DNA multi-site fusion reference substance shows a relatively consistent detection condition with the common DNA fusion reference substance in the market when the fusion gene of the conventional sites is detected. However, in tables 8-9 of example 10, the DNA multi-site fusion reference of the present invention shows that the capture efficiency of different sites is different even at the same sequencing depth when fusion genes such as NTRK 2-QKI-14 and ROS 1-QKI-80 are missed in the detection products selected by us. The detection capability of the whole detection product on the fusion site is judged only by the detection results of one or two sites, so that the problem of detection omission or false positive can occur due to over-high or over-low threshold value.
In addition, there is a certain range of fluctuation in the detection capability of the detection product with respect to the fusion of the ALK, ROS1, and other genes, which occurs at different positions within each gene. Therefore, the DNA multi-site fusion reference product covers different exon regions of a plurality of genes, and can accurately evaluate a plurality of sites of the genes such as ALK, ROS1 and the like, so that the reliability of the detection result is ensured, the detection capability of the fusion gene detection product can be evaluated from the whole level, the actual performance of the fusion gene detection product can be better reflected, and the effect of the reference product provided by the invention is better.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Fujian and Rui Gene science and technology Co., Ltd
<120> preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence
<400> 1
gcagcatgtc aagatcacag att 23
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
cctccttctg catggtattc tttct 25
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<400> 3
ctctccctcc ctccaggaa 19
<210> 4
<211> 15
<212> DNA
<213> Artificial sequence
<400> 4
caggcggcac acgtg 15
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence
<400> 5
gcatactgct gacagttaaa tttag 25
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
ccattgtggg tatttcagag 20
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence
<400> 7
tctaggattc agggtcctg 19
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
ccattgtggg tatttcagag 20
<210> 9
<211> 16
<212> DNA
<213> Artificial sequence
<400> 9
agtttggcca gcccaa 16
<210> 10
<211> 16
<212> DNA
<213> Artificial sequence
<400> 10
agtttggccc gcccaa 16
<210> 11
<211> 15
<212> DNA
<213> Artificial sequence
<400> 11
atggccagcg tggac 15
<210> 12
<211> 17
<212> DNA
<213> Artificial sequence
<400> 12
tgatggccat cgtggac 17
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence
<400> 13
agttgaagca caggctggat t 21
<210> 14
<211> 18
<212> DNA
<213> Artificial sequence
<400> 14
ccaccctctg gattactt 18
Claims (10)
1. The high-throughput sequencing method is applied to preparation or detection of a fusion gene positive fragment library or preparation or detection of a fusion gene detection reference.
2. A preparation or detection method of a fusion gene positive fragment library is characterized in that the preparation or detection method comprises the steps of detecting the quantity ratio of positive fragments in the fusion gene positive fragment library obtained by mixing different positive fragments by adopting a high-throughput sequencing method;
the preparation method of the fusion gene positive fragment library also comprises the step of adjusting the number of each positive fragment in the fusion gene positive fragment library.
3. The preparation or detection method of claim 2, wherein the step of detecting by the high throughput sequencing method comprises performing a homogeneity evaluation on each positive fragment in the fusion gene positive fragment library by a high throughput sequencing method after each positive fragment is subjected to the breaking, repairing, connecting and purifying treatment in sequence;
preferably, the homogeneity evaluation comprises evaluating the copy number of each positive fragment in the pool of fusion gene positive fragments;
preferably, the copy number of each positive fragment is obtained by detecting the sequence depth.
4. A pool of fusion gene positive fragments obtained by the production method according to claim 2 or claim 3;
preferably, each positive fragment in the fusion gene positive fragment library is mixed in an equimolar way;
preferably, the total concentration of the fusion gene positive fragments in the fusion gene positive fragment library is 2-10 ng/mu L.
And 5, application of ddPCR in preparation or detection of fusion gene detection reference substances.
6. A preparation or detection method of a fusion gene detection reference substance is characterized in that the preparation or detection method comprises the steps of detecting the mutation frequency of each positive fragment in the fusion gene detection reference substance by ddPCR (polymerase chain reaction), wherein the fusion gene detection reference substance comprises a fusion gene detection reference substance obtained by diluting a mixture of a marker locus positive fragment and a fusion gene positive fragment by negative DNA (deoxyribonucleic acid); the preparation or detection method further comprises the step of detecting the quantity ratio of the positive fragments in the fusion gene detection reference substance by adopting a high-throughput sequencing method;
preferably, the number of marker site positive fragments is at least 2.
7. A fused gene detection reference obtained by the production method according to claim 6;
preferably, the fusion gene detection reference comprises a tumor tissue reference, and the source of negative DNA of the tumor tissue reference comprises a wild-type GM12878 cell line;
preferably, the negative DNA of the tumor tissue reference is derived from gDNA of a wild-type GM12878 cell line;
preferably, the tumor tissue reference comprises at least 3 dilution gradients;
preferably, the dilution gradient of the tumor tissue reference comprises 5%, 2% and 1% of total moles of fusion gene positive fragments as a percentage of total moles of DNA;
preferably, the fusion gene detection reference comprises a plasma reference obtained by ultrasonically cutting the fusion gene positive fragment, and negative DNA of the plasma reference comprises plasma negative DNA;
preferably, the plasma reference dilution gradient comprises 1%, 0.5% and 0.2% fusion gene positive fragments as a percentage of total DNA moles.
8. The method for preparing or detecting according to claim 6 or the fusion gene detection reference according to claim 7, wherein the marker site positive fragments comprise SNP positive fragments;
preferably, the number of SNP positive fragments is 2, namely positive fragments 81 and 82.
9. The method for preparing the fusion gene positive fragment library according to claim 2 or 3, the fusion gene positive fragment library according to claim 4, the method for preparing the fusion gene detection reference according to claim 6, or the fusion gene positive fragments selected for the fusion gene detection reference according to claim 7 include at least 11 fusion gene positive fragments shown as positive fragments 1 to 80, the intervals between the corresponding gene loci of different fusion gene positive fragments are greater than 300bp, and the genome version of the fusion gene sequence origin gene reference shown as the positive fragments 1 to 80 is feb.2009(GRCh37/hg 19).
10. Use of the fused gene assay reference obtained by the production method according to claim 6 or the fused gene assay reference according to claim 7 in any one of (A) to (E):
(A) as a reference for DNA fusion gene detection;
(B) evaluating DNA fusion gene detection products based on high-throughput sequencing;
(C) calibrating the detection result of the DNA fusion gene;
(D) optimizing a DNA fusion gene detection product or a detection method system;
(E) tumor panel stability was verified and optimized.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110453841.8A CN113106149B (en) | 2021-04-26 | 2021-04-26 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110453841.8A CN113106149B (en) | 2021-04-26 | 2021-04-26 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113106149A true CN113106149A (en) | 2021-07-13 |
| CN113106149B CN113106149B (en) | 2023-01-17 |
Family
ID=76721821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110453841.8A Active CN113106149B (en) | 2021-04-26 | 2021-04-26 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113106149B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134217A (en) * | 2021-11-10 | 2022-03-04 | 上海思路迪生物医学科技有限公司 | Method, system and equipment for preparing detection limit sample and enterprise reference product |
| CN114395619A (en) * | 2021-12-29 | 2022-04-26 | 福建和瑞基因科技有限公司 | High-throughput sequencing method and internal reference quality control product |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094074A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 |
| CN106282163A (en) * | 2016-08-15 | 2017-01-04 | 天津诺禾医学检验所有限公司 | Construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection |
| WO2017062970A1 (en) * | 2015-10-10 | 2017-04-13 | Guardant Health, Inc. | Methods and applications of gene fusion detection in cell-free dna analysis |
| CN108085391A (en) * | 2017-12-28 | 2018-05-29 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation |
| CN108728516A (en) * | 2018-06-22 | 2018-11-02 | 安徽鼎晶生物科技有限公司 | A kind of preparation method of tumor sample sequencing reference material |
| CN108728492A (en) * | 2017-04-19 | 2018-11-02 | 辽宁琦润生物科技有限公司 | A kind of preparation method of gene mutation and fusion positive reference product |
| WO2018215497A1 (en) * | 2017-05-25 | 2018-11-29 | Koninklijke Philips N.V. | System and method for detecting gene fusion |
| CN109837275A (en) * | 2019-01-08 | 2019-06-04 | 大连医科大学附属第二医院 | A kind of preparation method of fusion positive control standard items |
| CN110438230A (en) * | 2019-08-19 | 2019-11-12 | 南京科佰生物科技有限公司 | NTRK fusion mutation standard items and its preparation method and application |
| CN111235225A (en) * | 2020-02-27 | 2020-06-05 | 菁良基因科技(深圳)有限公司 | Paraffin Embedding (FFPE) reference substance for detecting fusion gene RNA expression and preparation method and application thereof |
| CN112164423A (en) * | 2020-10-14 | 2021-01-01 | 深圳吉因加医学检验实验室 | Fusion gene detection method, device and storage medium based on RNAseq data |
-
2021
- 2021-04-26 CN CN202110453841.8A patent/CN113106149B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094074A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 |
| WO2017062970A1 (en) * | 2015-10-10 | 2017-04-13 | Guardant Health, Inc. | Methods and applications of gene fusion detection in cell-free dna analysis |
| CN106282163A (en) * | 2016-08-15 | 2017-01-04 | 天津诺禾医学检验所有限公司 | Construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection |
| CN108728492A (en) * | 2017-04-19 | 2018-11-02 | 辽宁琦润生物科技有限公司 | A kind of preparation method of gene mutation and fusion positive reference product |
| WO2018215497A1 (en) * | 2017-05-25 | 2018-11-29 | Koninklijke Philips N.V. | System and method for detecting gene fusion |
| CN108085391A (en) * | 2017-12-28 | 2018-05-29 | 广州市金圻睿生物科技有限责任公司 | A kind of RNA quality-control products for the lung cancer fusion detection kit for stablizing preservation |
| CN108728516A (en) * | 2018-06-22 | 2018-11-02 | 安徽鼎晶生物科技有限公司 | A kind of preparation method of tumor sample sequencing reference material |
| CN109837275A (en) * | 2019-01-08 | 2019-06-04 | 大连医科大学附属第二医院 | A kind of preparation method of fusion positive control standard items |
| CN110438230A (en) * | 2019-08-19 | 2019-11-12 | 南京科佰生物科技有限公司 | NTRK fusion mutation standard items and its preparation method and application |
| CN111235225A (en) * | 2020-02-27 | 2020-06-05 | 菁良基因科技(深圳)有限公司 | Paraffin Embedding (FFPE) reference substance for detecting fusion gene RNA expression and preparation method and application thereof |
| CN112164423A (en) * | 2020-10-14 | 2021-01-01 | 深圳吉因加医学检验实验室 | Fusion gene detection method, device and storage medium based on RNAseq data |
Non-Patent Citations (4)
| Title |
|---|
| EDDY N DE BOER等: "Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing", 《CLINICAL CHEMISTRY》 * |
| QINGGUO WANG等: "Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives", 《BRIEFINGS IN BIOINFORMATICS》 * |
| 张文新等: "肿瘤基因突变检测的标准化研究", 《中国医药生物技术》 * |
| 曲守方等: "BCR-ABL融合基因检测试剂盒质控品的建立", 《药物分析杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134217A (en) * | 2021-11-10 | 2022-03-04 | 上海思路迪生物医学科技有限公司 | Method, system and equipment for preparing detection limit sample and enterprise reference product |
| CN114134217B (en) * | 2021-11-10 | 2024-06-11 | 上海思路迪生物医学科技有限公司 | Method, system and equipment for preparing detection limit sample and enterprise reference |
| CN114395619A (en) * | 2021-12-29 | 2022-04-26 | 福建和瑞基因科技有限公司 | High-throughput sequencing method and internal reference quality control product |
| CN114395619B (en) * | 2021-12-29 | 2024-04-30 | 福建和瑞基因科技有限公司 | High-throughput sequencing method and internal reference quality control product |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113106149B (en) | 2023-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113106149B (en) | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance | |
| CN110628880B (en) | Method for detecting gene variation by synchronously using messenger RNA and genome DNA template | |
| US9434941B2 (en) | Method for producing circular DNA formed from single-molecule DNA | |
| CN113462784A (en) | Method for constructing target set for homologous recombination repair defect detection | |
| US20230272475A1 (en) | A method for detecting the mutation and methylation of tumor-specific genes in ctdna | |
| US20220177874A1 (en) | Methods for library preparation to enrich informative dna fragments using enzymatic digestion | |
| EP3704267A1 (en) | Cancer detection, classification, prognostication, therapy prediction and therapy monitoring using methylome analysis | |
| CN111334505B (en) | Standard substance for pan-tumor gene detection and preparation method and application thereof | |
| WO2019076018A1 (en) | Method for constructing amplicon library for detecting low-frequency mutation of target gene | |
| WO2023216707A1 (en) | Universal preclinical bio-distribution detection kit for nk cell therapy products | |
| CN111041069A (en) | High-throughput sequencing library construction method for low-initial-quantity DNA sample and application thereof | |
| CN116355987A (en) | Method for producing a reference for gene mutation, reference for gene mutation and use thereof | |
| CN115369159A (en) | Ultralow frequency mutation detection method based on double-end sequencing overlapping fragment and DNA double-strand complementary fragment | |
| US20220127601A1 (en) | Method of determining the origin of nucleic acids in a mixed sample | |
| CN112251512B (en) | Target genome for gene detection of non-small cell lung cancer patient and related evaluation method, application and kit | |
| CN108624686A (en) | A kind of probe library, detection method and the kit of detection BRCA1/2 mutation | |
| EP4281583A1 (en) | Heatrich-bs: heat enrichment of cpg-rich regions for bisulfite sequencing | |
| WO2021072374A1 (en) | Use of simultaneous marker detection for assessing difuse glioma and responsiveness to treatment | |
| CN112280864A (en) | Thyroid polygene joint detection kit | |
| KR20190078715A (en) | Next generation sequencing (ngs)-based hybrid diagnostic panel for analyzing variation of cancer gene and anticancer drug-related gene | |
| US20240102089A1 (en) | Method for Evaluating Adapter Ligation Efficiency in Sequencing of DNA Sample | |
| US20230227915A1 (en) | METHOD FOR DETECTING CANCER USING 5-HYDROXYMETHYLCYTOSINE (5-hmC) | |
| Zhang et al. | Accurate somatic variant calling performance using Avidity Sequencing with Burning Rock OncoScreenTM Plus panel | |
| Jones et al. | Plasma ctDNA enables early detection of temozolomide resistance mutations in glioma | |
| CN118006770A (en) | Detection of cancer using positive and negative strand differences of free DNA molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |