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WO2019173991A1 - Marqueur de lymphome malin et son application - Google Patents

Marqueur de lymphome malin et son application Download PDF

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Publication number
WO2019173991A1
WO2019173991A1 PCT/CN2018/079061 CN2018079061W WO2019173991A1 WO 2019173991 A1 WO2019173991 A1 WO 2019173991A1 CN 2018079061 W CN2018079061 W CN 2018079061W WO 2019173991 A1 WO2019173991 A1 WO 2019173991A1
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mutation
sequencing
probe
candidate
sequence
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PCT/CN2018/079061
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English (en)
Chinese (zh)
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潘嫱
叶晓飞
苏红
刘栋兵
任伟成
吴逵
朱师达
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深圳华大生命科学研究院
潘嫱
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Application filed by 深圳华大生命科学研究院, 潘嫱 filed Critical 深圳华大生命科学研究院
Priority to PCT/CN2018/079061 priority Critical patent/WO2019173991A1/fr
Priority to CN201880083693.1A priority patent/CN111655868A/zh
Publication of WO2019173991A1 publication Critical patent/WO2019173991A1/fr

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    • 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

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  • the invention relates to the field of gene sequencing and medical detection, in particular to a malignant lymphoma marker and application thereof, in particular to a malignant lymphoma marker and a probe and a chip for detecting the marker, and constructing a malignant sample to be tested
  • a method and system for determining a genetic mutation in a malignant lymphoma in a sample to be tested by a method for detecting a sequencing library of lymphoma A method and system for determining a genetic mutation in a malignant lymphoma in a sample to be tested by a method for detecting a sequencing library of lymphoma.
  • Malignant lymphoma is a type of systemic disease that is closely related to the functional status of the body's immune system. It is different from other solid malignant tumors and different from blood tumors. It includes a disease of Hodgkin's lymphoma and a group of diseases of non-Hodgkin's lymphoma. The clinical manifestations are complicated by the type of pathology, stage and invasion. Currently, multiple FDA-approved molecularly targeted drugs are available for malignant lymphomas such as Ibrutinib (BTK) and Idelalisib (PI3K delta), so accurate and timely detection of malignant lymphoma gene mutations is significant for clinical diagnosis and treatment. The meaning.
  • BTK Ibrutinib
  • PI3K delta Idelalisib
  • an object of the present invention is to provide a malignant lymphoma marker and a probe and a chip for detecting the same, and a method for constructing a sequencing library of a malignant lymphoma detection sample to determine a malignant lymphocyte in a sample to be tested. Methods and systems for gene mutations in tumors.
  • the invention provides a malignant lymphoma marker comprising the genes in the following table:
  • the present invention selects 212 genes which are highly associated with malignant lymphoma as markers related to malignant lymphoma, and the present invention is stronger than the technique based on detection of all genes associated with multiple cancers at one time. Targeted, and the detection range is smaller, the detection cost is lower, and the efficiency can be significantly improved while improving the efficiency.
  • the malignant lymphoma is a diffuse large B-cell lymphoma.
  • the invention provides a probe for a malignant lymphoma.
  • the probe is designed for all exon regions in the marker described in the above table and the junction region of the exon and the intron, the probe specifically recognizing the above The at least one portion of the malignant lymphoma marker coding region, and the probe satisfies at least one selected from the group consisting of:
  • the length of the probe is 75-85 bp, preferably 81 bp;
  • the probe specifically recognizes a sequence from 10 bp upstream to 10 bp downstream of the marker coding region described in the above Examples;
  • the melting temperature of the probe and the target sequence is 60-10 degrees Celsius, preferably 80 degrees Celsius;
  • the probe does not comprise a hairpin structure
  • the window sliding size when the probe is selected is 10 bp.
  • the present invention provides a gene chip.
  • the gene chip comprises a probe and a support, the probe being located on the surface of the support, the probe being the probe described in the above embodiments.
  • the gene chip may further add the following technical features:
  • the gene chip is a liquid phase chip and the support is a microsphere containing different fluorescent labels.
  • the present invention provides a method for constructing a sequencing library of a malignant lymphoma detection sample to be tested, comprising: enriching a target sequence of a sample to be tested, the target sequence being as described in the above table A malignant lymphoma marker, and the enriched target sequence constitutes the sequencing library for malignant lymphoma detection.
  • the method may further add the following technical features:
  • the target sequence of the sample to be tested is subjected to hybridization capture using the probe described in the above embodiment or the gene chip described in the above embodiment, thereby achieving the enrichment.
  • the method further comprises: sequencing the sequencing library for malignant lymphoma detection to obtain a sequencing sequence.
  • the sequencing library for malignant lymphoma detection is sequenced using a BGISeq-500 sequencing platform.
  • the sequencing sequence has a sequencing depth of 400 ⁇ or more, and the coverage of the sequencing sequence reaches 99% or more.
  • the raw data amount of the sequencing sequence is above 3Gb.
  • the present invention provides a method of determining a gene mutation of a malignant lymphoma in a sample to be tested. According to an embodiment of the invention, the method comprises:
  • the sequencing sequence is aligned to a reference genome, and mutation detection is performed to obtain candidate mutation data;
  • the potential mutation data is annotated to obtain target mutation data.
  • the above method for determining a gene mutation for a malignant lymphoma may further include the following technical features:
  • the reference genome is the human reference genome hg19.
  • the mutation detection is performed using VarScan software.
  • screening the candidate mutation data comprises: filtering out low quality, low coverage, candidate mutations located at both ends of the repeat region and the sequence, and having chain bias, wherein A low-mass candidate mutation refers to a candidate mutation having a base mass value of less than 20 or a ratio of less than 30, and the candidate mutation of the low coverage refers to a candidate mutation having a minimum support number of less than 3.
  • the annotation is performed by using ANNOVA software
  • the polymorphic site is filtered out by using a population mutation database
  • the benign mutation is filtered out by using the pathogenic mutation database.
  • the population mutation database is selected from at least one of a thousand human genome database, an ExAc database, and an Esp6500 database.
  • the disease-causing mutation database is ClinVar.
  • the method further comprises:
  • the sequencing sequence Prior to said mutation detection, the sequencing sequence is quality controlled to filter out low quality and linker contamination sequences, and the filtered sequences are then aligned to the reference genome.
  • the present invention provides a system for determining a gene mutation of a malignant lymphoma in a sample to be tested.
  • the system comprises:
  • target region library construction unit wherein the target region library construction unit is based on the marker described in the above embodiment as a target region, thereby constructing a target region library
  • the sequencing unit is connected to the target region library building unit, and the sequencing unit detects the target region library to obtain a sequencing sequence;
  • a candidate mutation determining unit wherein the candidate mutation determining unit is connected to the sequencing unit, wherein the candidate mutation determining unit is configured to compare the sequencing sequence in the target region library to the reference genome, and perform mutation detection to obtain candidate mutation data;
  • a potential mutation determining unit the potential mutation determining unit being connected to the candidate mutation determining unit, wherein the potential mutation determining unit is configured to screen the candidate mutation data to obtain potential mutation data;
  • the target mutation determining unit is connected to the potential mutation determining unit, and the target mutation determining unit is configured to annotate the potential mutation data to obtain target mutation data.
  • the system for determining a gene mutation for a malignant lymphoma may further include the following technical features:
  • the reference genome is a human reference genome hg.
  • the mutation detection is performed using VarScan software.
  • screening the candidate mutation data comprises: filtering out low quality candidate mutations, low coverage candidate mutations, candidate mutations at both ends of the repeat region and the sequence, and having a chain bias
  • candidate mutations in which the low-mass candidate mutation refers to a candidate mutation having a base mass value of less than 20 or a specific mass value of less than 30, and the low-coverage candidate mutation refers to a candidate mutation having a minimum support number of less than 3.
  • the annotation is performed by using ANNOVA software, the polymorphic site is filtered out by using a population mutation database, and the benign mutation is filtered out by using the pathogenic mutation database.
  • the population mutation database is selected from at least one of a thousand human genome database, an ExAc database, and an Esp6500 database.
  • the disease-causing mutation database is ClinVar.
  • system further comprises:
  • a quality control unit the quality control unit being coupled to the sequencing unit, the quality control unit for performing quality control on the sequencing sequence prior to the detecting of the mutation, thereby filtering out low quality and joint contamination sequences, and then filtering The filtered sequences are aligned to the reference genome.
  • the invention provides the use of a combination of 212 markers in the above table for the preparation of a reagent for the detection and/or determination of a mutation in a malignant lymphoma gene.
  • the invention provides the use of the combination of 212 markers in the above table for the detection and/or determination of genetic mutations in malignant lymphoma.
  • the present invention enriches 212 specific malignant lymphoma specific target genes, and then uses high-throughput sequencing means for detecting and determining a mutant gene associated with malignant lymphoma. It can be quickly and effectively used to detect single base substitutions, single base/multibase insertions or deletions in target sequences, and large fragment deletion/amplification mutation types, which can meet the high-efficiency and comprehensive detection of common malignant lymphoma gene mutations. .
  • the method of detection by means of the BGISEQ-500 second-generation sequencing platform has the advantages of wide application range, high efficiency, comprehensiveness and easy operation, and realizes rapid and efficient determination of genes related to malignant lymphoma.
  • FIG. 1 is a schematic diagram of a system for determining genetic mutations in a malignant lymphoma, in accordance with an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of a system for determining genetic mutations in a malignant lymphoma, in accordance with an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of obtaining a target mutation by analyzing a sequencing sequence according to an embodiment of the present invention.
  • the method for target sequence capture and high-throughput sequencing of malignant lymphoma genes as described in the present invention is designed based on the needs of the gene mutation detection technology for malignant lymphoma.
  • the present invention targets all exon regions and exon-intron junction regions of common malignant lymphoma genes (212 genes shown in Table 1) as target capture regions, and designs probes capable of simultaneously capturing all target sequence regions. Combine and then customize the liquid phase chip (produced by Huada Gene) and combine the BGISEQ-second generation high-throughput sequencing technology and information analysis technology to sequence all captured target sequences and different types of mutation information.
  • the invention has the advantages of wide application range, high efficiency, comprehensiveness, easy operation, and the like, and detects single base substitution, single base/multibase insertion or deletion, and large fragment deletion/amplification in the target sequence, and satisfies malignancy. Efficient, comprehensive detection of lymphoma gene mutations.
  • the inventors of the present invention collected and analyzed a plurality of genes associated with malignant lymphoma by conducting research and analysis, and finally determined 212 gene combinations related to malignant lymphoma according to their correlation and pathogenicity (eg, 1)), as a marker for identifying malignant lymphoma, can also use these markers as target regions to enrich them, and can effectively detect and/or identify genetic mutations associated with malignant lymphoma, including However, it is not limited to single base substitution, single base/multibase insertion or deletion, and large fragment deletion/amplification, so that it can satisfy the high-efficiency and comprehensive detection of malignant lymphoma gene mutations. The sensitivity is over 93%.
  • Tables 2 and 3 list the names of the malignant lymphoma cancer genes and their corresponding malignant lymphoma names, respectively. Based on a series of theoretical studies and experimental verification work, the inventors discovered and demonstrated the correlation between the 212 genes in the above table, and concluded that the effective detection of malignant lymphoma can be achieved by using this group of genes, and With a single gene or other combination of genes as markers, the test results are more accurate, reliable, and reproducible.
  • this group of genes is involved in important pathogenic signaling pathways of lymphoma, such as BCR, chromatin modification, apoptosis and cell cycle regulation, immunosuppression, and Notch.
  • This group of genes has broad and comprehensive advantages in the field of lymphoma cancer gene detection.
  • these genes are also listed separately in the literature with high impact factors, and, to date, no report has been made to use the combination of these 212 genes as a marker for malignant lymphoma.
  • KLF2 gene, ZFP36L1 gene, and TMSB4X gene are the first inventors to discover that the frequency of mutations in Asian ethnic groups is significantly higher than that of Caucasians. gene.
  • genes associated with malignant lymphoma are associated with diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, especially with diffuse large B-cell lymphoma.
  • Important in Table 1 refers to an important pathogenic signaling pathway present in lymphoma.
  • the inventors designed a probe and gene chip that can be used for malignant lymphoma.
  • the probe is designed with all exon regions and exon and intron junction regions of the 212 target cancer genes as the total target region, and the probe specifically recognizes the above 212 markers.
  • the probe satisfies at least one selected from the group consisting of: (1) the probe has a length of 75-85 bp, preferably 81 bp; (2) the probe specifically recognizes this 212 sequences between 10 bp and 10 bp downstream of the marker coding region; (3) probes that specifically recognize regions with GC content higher than 0.6 and below 0.3, multiplier greater than 2; (4) The melting temperature of the probe to the target sequence is 60-10 degrees Celsius, preferably 80 degrees Celsius; (5) the probe does not comprise a hairpin structure; (6) the probe matches at most 2 sites on the reference genome; (7) The window sliding size when the probe is selected is 10 bp.
  • the probes for malignant lymphoma designed according to the above principles contain a total of 32779 probes, each of which has a length of 81 bp, and each of which contains a 16 bp and 15 bp tag sequence, and the sequence of the two tag sequences.
  • the two tag sequences, GAAGCGAGGATCAACT (SEQ ID NO: 1) and CATTGCGTGAACCGA (SEQ ID NO: 2), respectively, are the restriction sites and transcription sites, and both ends are used to design PCR primers, and the transcription sites are simultaneously Used for transcription and functions as an RNA probe.
  • the inventors have also devised a gene chip comprising a probe and a support, the probe being located on the surface of the support.
  • the gene chip may be designed as a liquid phase chip, and the support is a microsphere containing different fluorescent labels.
  • a sequencing library for malignant lymphoma can be constructed, and on the basis of this, the sequencing is performed.
  • Bioinformatics analysis of the library can effectively detect and/or identify genetic mutations associated with malignant lymphoma, including but not limited to single base substitutions, single base/multibase insertions or deletions, and large fragment deletions/amplifications
  • the type of mutation can meet the high-efficiency and comprehensive detection of gene mutations in malignant lymphoma. It has been proved by experiments that the sensitivity is over 93%.
  • the method for determining a gene mutation of a malignant lymphoma in a sample to be tested comprises: enriching a target sequence of a sample to be tested, wherein the target sequence is a combination of 212 malignant lymphoma markers,
  • the obtained target sequence constitutes the sequencing library for detection of malignant lymphoma;
  • the sequencing library of the malignant lymphoma detection is sequenced to obtain a sequencing sequence;
  • the sequencing sequence is aligned to a reference genome for mutation Detecting, obtaining candidate mutation data; screening the candidate mutation data to obtain potential mutation data; annotating the potential mutation data to obtain target mutation data.
  • the sample to be tested in the present invention may be derived from a tissue sample.
  • the method for determining a gene mutation of a malignant lymphoma in a sample to be tested can also be expressed as a method for detecting and/or determining a gene mutation for a malignant lymphoma, which is not a method for diagnosing a disease.
  • the mutation results detected by the present invention can only indicate that the cancer tissue of the relevant individual carries a consistent cancer-driven gene mutation, and in practice, it is also necessary to combine the clinical results to confirm the individual's disease.
  • target region DNA enrichment method based on multiplex PCR technology (such as Thermo Fisher Scientific AmpliSeq technology) and Target region DNA enrichment methods based on probe hybridization techniques (such as Agilent's SureSelect technology, and Nimble's SeqCap EZ technology).
  • Illumina's Hiseq/Miseq/NextSeq, Thermo Fisher Scientific's Ion Proton/Ion PGM, and BGI SEQ-500 of the BGI gene can be used.
  • the sequencing sequence is obtained by sequencing using a BGISeq-500 sequencing platform.
  • High-throughput sequencing using a self-developed sequencer from Huada has stronger compatibility and better sequencing results.
  • the original data volume of the constructed sequencing library reaches 3 Gb or more
  • the target region has a sequencing depth of 400 ⁇ or more
  • the target region coverage reaches 99% or more.
  • the sequencing depth refers to the ratio of the total number of bases (bp) and the genome size (Genome) obtained by sequencing, reflecting the average number of times a single base on the tested genome is sequenced.
  • Sequencing coverage refers to the proportion of sequences obtained by sequencing to the entire genome.
  • the candidate mutation data is screened, including screening for removal of low-quality, low-coverage, candidate mutation data located at the ends of the repeat region and the sequence, and having strand bias.
  • the low-quality candidate mutation refers to a sequence having a base mass value of less than 20 (base quality ⁇ 20) or a pair of quality values (mapping quality ⁇ 30), and a low-coverage candidate mutation refers to a minimum support number of less than 3 ( Candidate mutations for minimal support depth ⁇ 3).
  • Candidate mutations with strand bias refer to candidate mutations that occur only on one strand.
  • the present invention is based on a combination of 212 genes associated with a lymphoma associated with the discovery of the inventors, and a combination of the 212 specific genes as a target gene, and a system for determining a gene mutation of a malignant lymphoma in a sample to be tested is designed.
  • the system for determining a gene mutation of a malignant lymphoma in a sample to be tested according to the present invention can also be understood as a system for detecting a gene mutation of a malignant lymphoma in a sample to be tested, and is used for detecting and determining a malignant sample in a sample to be tested.
  • the present invention provides a system for determining a gene mutation of a malignant lymphoma in a sample to be tested, as shown in FIG. 1, the system comprising: a target region library building unit, and the target region library construction The unit constructs a library of target regions based on the combination of markers in the present invention 212 as a target region; a sequencing unit, the sequencing unit is connected to the target region library building unit, and the sequencing unit detects the target region library so that Obtaining a sequencing sequence; a candidate mutation determining unit, wherein the candidate mutation determining unit is connected to the sequencing unit, wherein the candidate mutation determining unit is configured to compare the sequencing sequence in the target region library to the reference genome, and perform mutation detection to obtain Candidate mutation data; a potential mutation determining unit, the potential mutation determining unit being linked to the candidate mutation determining unit, wherein the potential mutation determining unit is configured to screen the candidate mutation data to obtain potential mutation data; Unit, the target mutation determining unit and the potential The mutation determining unit is
  • the system for determining a gene mutation of a malignant lymphoma in a sample to be tested may also be as shown in FIG. 2, the system comprising: a target region library building unit, wherein the target region library building unit is based on The marker combination in the invention 212 is used as a target region to construct a target region library; the sequencing unit is connected to the target region library construction unit, and the sequencing unit detects the target region library; the quality control unit, The quality control unit is coupled to the sequencing unit, and the quality control unit is configured to perform quality control on the sequencing sequence before the mutation detection, thereby filtering out low quality and joint contamination sequences, and then filtering the filtered Aligning a sequence to the reference genome; a candidate mutation determining unit, the candidate mutation determining unit being coupled to a quality control unit, the candidate mutation determining unit for aligning the filtered sequence to the reference genome , performing mutation detection to obtain candidate mutation data; potential mutation determining unit, the latent a mutation determining unit is coupled to the candidate mutation
  • the length of the probe is 81 bp
  • the probe specifically recognizes a sequence of from 10 bp upstream to 10 bp downstream of 212 of the marker coding regions in Table 1;
  • the melting temperature of the probe and the target sequence is 60-10 degrees Celsius, preferably 80 degrees Celsius;
  • the probe does not comprise a hairpin structure
  • the window sliding size when the probe is selected is 10 bp.
  • the finally obtained target region probe sequence contains 32779 probes, each of which has a length of 81 bp, and each of which contains a 16 bp and 15 bp tag sequence, and the sequence of the two tag sequences is GAAGCGAGGATCAACT (SEQ ID NO). : 1) and CATTGCGTGAACCGA (SEQ ID NO: 2).
  • the two tag sequences are respectively an enzyme cleavage site and a transcription site, and both ends are used to design PCR primers, and the transcription site is used for transcription and functions as an RNA probe.
  • KLF2 gene probe sequence (SEQ ID NO: 3)
  • KLF2 gene probe sequence (SEQ ID NO: 4)
  • KLF2 gene probe sequence (SEQ ID NO: 5)
  • KLF2 gene probe sequence (SEQ ID NO: 6)
  • KLF2 gene probe sequence (SEQ ID NO: 7)
  • ZFP36L1 gene probe sequence (SEQ ID NO: 8)
  • ZFP36L1 gene probe sequence (SEQ ID NO: 9)
  • ZFP36L1 gene probe sequence (SEQ ID NO: 10)
  • TMSB4X gene probe sequence (SEQ ID NO: 13)
  • TMSB4X gene probe sequence (SEQ ID NO: 14)
  • TMSB4X gene probe sequence (SEQ ID NO: 15)
  • TMSB4X gene probe sequence (SEQ ID NO: 16)
  • TMSB4X gene probe sequence (SEQ ID NO: 17)
  • the liquid phase chip is prepared by using polysphere microspheres having a diameter of about 5.6 ⁇ m, a carboxyl group on the surface, and red and orange dyes inside, according to the ratio of the two dyes. The difference can be divided into 100 kinds of microspheres, each with a number. Each microsphere has a specific spectral characteristic due to the difference in internal fluorescence ratio and can be specifically recognized by the laser. Different probe molecules are coated with different numbered microspheres to detect the target molecule in the sample, and the target molecule is then combined with the reporter molecule with fluorescence. The detection of the molecule of interest is then achieved by fluorescence detection.
  • the experimental samples used were 16 tissue samples clinically diagnosed as diffuse large B-cell lymphoma.
  • the specific experimental methods are as follows:
  • Genomic DNA was extracted from diffuse large B-cell lymphoma tissue samples using the QIAGEN DNA Tissue and Blood mini kit and using the QIAGEN DNA Tissue and Blood mini kit, as described in the kit's extraction instructions.
  • Fluorescence analyzer to detect DNA concentration, the required concentration is greater than 5ng / ⁇ L, the volume is greater than 30 ⁇ L, and in principle, the DNA yield of each sample is ⁇ 2 ⁇ g, then the DNA is detected by electrophoresis and its degradation degree, which is not suitable for the seriously degraded sample.
  • the library wherein the electrophoresis conditions were: 1% agarose gel, electrophoresis voltage 4 V/cm, electrophoresis time 45 min. The results of agarose gel electrophoresis showed that the DNA of all samples was intact and substantially free of degradation.
  • genomic DNA 100 ng was taken and randomly interrupted by enzyme digestion using a DNA interrupter, and the terminal repair and A were simultaneously performed; followed by ligation and purification, PCR amplification, obtaining a pre-hybrid library, and using the Agient 2100 bioanalyzer.
  • the second fragment is screened to obtain a length fragment of 150-500 bp; then the PCR product is subjected to target region hybridization capture using a liquid phase capture chip, and the target DNA is eluted from the probe by an elution reagent to obtain a desired target DNA. After that, PCR amplification is performed.
  • the resulting product was cyclized to construct a library captured in the region of interest, wherein the yield of the hybrid library obtained was greater than 160 ng.
  • liquid phase capture chip used was prepared as in Example 1.
  • the library DNA after the quality control was subjected to sequencing on the basis of the operation instructions of BGISeq-500 sequencing.
  • the obtained raw data amount of each sample reached more than 3Gb, the average sequencing depth of the target area reached 400 ⁇ , and the target area coverage was over 99%.
  • the quality of the sequencing data of 16 samples is shown in Table 4 below.
  • quality control is performed on the prepared reads, thereby removing sequences whose sequencing quality is not in conformity with the requirements and sequencing of the junction contamination, and obtaining a clean sequence (ie, the filtered sequence).
  • the filtered sequence was then aligned to the human reference genome Hg19 (http://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips/) using bwa (Burrows-Wheeler Aligner) software to obtain alignment results.
  • VarScan software to detect mutations, obtain candidate mutations, and perform initial filtering on candidate mutation results, filtering out low quality (base quality ⁇ 20 or mapping quality ⁇ 30), low coverage (minimal support depth ⁇ 3), and repeating The region and the ends of the reads, with strand-biased mutation sites, eventually yielded a list of potential mutations.
  • a list of potential mutations obtained is annotated with ANNOVA software, excluding synonymous mutations therein. Then use a population mutation database (such as the Thousand Genome Database (http://www.1000genomes.org), ExAC database and Esp6500 database) to filter the common polymorphic sites in the population. Using a pathogenic mutation database (such as ClinVar), the benign mutation is filtered out and the final mutation result is obtained, that is, the target mutation data is obtained.
  • the synonymous mutation is a neutral mutation. Due to the degenerate phenomenon of the genetic code of the organism, when the synonymous mutation occurs, the base is replaced, and a new codon is generated, but the new and old codons are encoded. The amino acid type remains unchanged, so this part of the mutation does not have any effect on the pathogenic condition.
  • the experimental results showed that 163 cancer mutation sites were detected by analyzing and filtering the sequencing data of 16 samples.
  • This example utilizes the same sample as in the second embodiment, and uses the hiseq2000 sequencing platform to construct a sequencing library corresponding to each sample according to its operation guide according to the method of whole genome sequencing, and according to the same method as step 4 in the second embodiment.
  • Example 2 Comparing the experimental results of Example 2 and Example 3, it can be seen that a total of 174 cancer mutations were detected in all 16 patients with diffuse large B-cell lymphoma using whole-genome sequencing, and the use was related to malignant lymphoma. Of the 212 target gene capture methods, a total of 163 cancer mutations were detected in the 174 cancer mutations. Comparing the two results, it was observed that mutations were made using 212 target gene captures associated with malignant lymphoma. Detection, compared to the whole genome sequencing for mutation detection, the overall sensitivity reached 93.7%. The detailed detection of each sample is shown in Table 5 below, including SNP mutation sites and insertion deletion variants (Indel mutations):
  • the correlation between the minimum allele frequency detected by the target gene capture and the minimum allele frequency detected by whole genome sequencing was as high as 0.8186 (r2, Pearson correlation coefficient) as shown in FIG.
  • the abscissa in Figure 4 represents the minimum allele frequency (MAF in WGS, Minor allele frequency in Whole-genome-sequencing) obtained by whole genome sequencing, and the ordinate represents the minimum obtained by target gene capture sequencing.
  • the terms “installation”, “connected”, “connected”, “fixed” and the like shall be understood broadly, and may be either a fixed connection or a detachable connection, unless explicitly stated and defined otherwise. Or in one piece; it may be a mechanical connection, or it may be an electrical connection or a communication with each other; it may be directly connected or indirectly connected through an intermediate medium, and may be an internal connection of two elements or an interaction relationship between two elements. Unless otherwise expressly defined. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood on a case-by-case basis.

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Abstract

L'invention concerne un marqueur de lymphome malin. Le marqueur de lymphome malin comprend 212 gènes au total, tels que AICDA et AKT1. L'invention concerne en outre une application du marqueur dans les domaines du séquençage génique et de la détection médicale.
PCT/CN2018/079061 2018-03-14 2018-03-14 Marqueur de lymphome malin et son application WO2019173991A1 (fr)

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