WO2016095093A1 - Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible - Google Patents

Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible Download PDF

Info

Publication number
WO2016095093A1
WO2016095093A1 PCT/CN2014/093871 CN2014093871W WO2016095093A1 WO 2016095093 A1 WO2016095093 A1 WO 2016095093A1 CN 2014093871 W CN2014093871 W CN 2014093871W WO 2016095093 A1 WO2016095093 A1 WO 2016095093A1
Authority
WO
WIPO (PCT)
Prior art keywords
reference sequence
region
genes
alignment
fragment
Prior art date
Application number
PCT/CN2014/093871
Other languages
English (en)
Chinese (zh)
Inventor
蔡宇航
陈希
钱朝阳
管彦芳
易鑫
朱红梅
杨玲
吴仁花
Original Assignee
天津华大基因科技有限公司
深圳华大基因科技有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 天津华大基因科技有限公司, 深圳华大基因科技有限公司 filed Critical 天津华大基因科技有限公司
Priority to PCT/CN2014/093871 priority Critical patent/WO2016095093A1/fr
Publication of WO2016095093A1 publication Critical patent/WO2016095093A1/fr

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

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to a target region variation detecting method, a target region variation detecting device, a tumor screening method, and a device.
  • Tumors are classified into benign tumors and malignant tumors. Malignant tumors are also called cancers. They are diseases caused by abnormal mechanisms controlling cell growth and proliferation. Cancer is one of the diseases that threaten human health. In addition to uncontrolled growth of cancer cells, cancer cells locally invade the surrounding normal tissues and even transfer to other parts of the body via the internal circulatory system or lymphatic system.
  • the present invention is directed to at least one of the technical problems existing in the prior art or at least one means.
  • the present invention provides a method of detecting a variation of a target region, the method comprising: (1) acquiring a nucleic acid in a sample to be tested, the nucleic acid being composed of a plurality of nucleic acid fragments, the nucleic acid fragment being derived from Broken genomic DNA and/or free DNA; (2) capturing the nucleic acid fragment using a kit to obtain a target region; (3) performing sequence determination on the target region to obtain sequencing data, the sequencing data being read by multiple Segment composition; (4) detecting a variation in the target region based on the sequencing data; wherein the kit comprises a probe capable of specifically recognizing the following predetermined regions: 547 genes in Table 1 The gene region of at least 10 genes in the gene.
  • the predetermined area is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 of the 547 genes.
  • the combination of gene regions that can be specifically recognized by the kit probe in the method of the present invention is obtained by the inventor through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are common tumor occurrence or developmental correlations. region.
  • the common tumors include lung cancer, colorectal cancer, stomach cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer, and liver cancer.
  • the related gene sequences of a plurality of common cancers can be obtained in one time, simply and conveniently, and high specificity, and the related gene sequences can be detected and analyzed, and the detection analysis results can be used for early screening of various common cancers. Check and judge the possibility and effect of artificially intervening early tumor intervention. At present, most cancers such as lung cancer, liver cancer, and gastric cancer have been diagnosed at the time of hospital pathological diagnosis, which delays the earlier treatment time and greatly reduces the possibility of cure.
  • the predetermined region is a gene region of 145 genes listed in Table 2 of the 547 genes.
  • the gene region of the 145 genes in Table 2 that the probe can specifically recognize is obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are associated with the development of lung cancer.
  • all lung cancer-related gene sequences can be obtained in a single, simple, convenient and highly specific manner, and the information obtained by detecting these gene sequences can assist in early screening of lung cancer. diagnosis.
  • the predetermined region is a gene region of 60 genes listed in Table 3 of the 547 genes.
  • the gene regions of the 60 genes of Table 3 that the probe can specifically recognize are obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are associated with the development of colorectal cancer.
  • all colorectal cancer-related gene sequences can be obtained in a single, simple, convenient and highly specific manner, and the information obtained by detecting these gene sequences can assist colorectal cancer. Early screening diagnosis.
  • the predetermined region is the gene region of the 43 genes listed in Table 4 of the 547 genes.
  • the gene regions of the 43 genes in Table 4 that the probe can specifically recognize are obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are related to the occurrence and development of gynecological reproductive tract tumors.
  • the genital tract tumors include ovarian cancer, endometrial cancer, and cervical cancer.
  • the probe has a length of from 25 to 300 nt, preferably from 50 to 250 nt, and more preferably from 80 nt to 120 nt.
  • the probe is obtained by first obtaining an initial probe set and then screening the initial probe set.
  • Obtaining the initial probe set includes: determining a reference sequence of the gene region, starting from one end of the reference sequence, sequentially acquiring a DNA fragment on the reference sequence to the other end of the reference sequence, wherein one DNA The fragment is an initial probe, and all of the DNA fragments constitute the initial probe set, the DNA fragments completely overlap, partially overlap or not overlap at all, and the initial probe set can cover the gene region at least once .
  • the reference sequence of the gene region can be obtained from the reference genome, for example, the corresponding gene region is obtained from the human reference genome HG19, and the corresponding gene region on all HG19 constitutes the reference sequence of the gene region, and HG19 can be NCBI database download.
  • the initial probe set is obtained by using an iterative algorithm design, including: determining a position of the gene region on a reference genome, and obtaining a reference sequence of the gene region, from the reference sequence
  • the first nucleotide begins to copy the reference sequence to obtain a first DNA fragment
  • the reference sequence is copied from the second nucleotide of the reference sequence to obtain a second DNA fragment, from which the reference sequence
  • the third nucleotide begins to copy the reference sequence to obtain a third DNA fragment, such that the subsequent DNA fragment is sequentially obtained until one end of the Nth DNA fragment exceeds the reference sequence, wherein one DNA fragment is an initial probe.
  • N is the total number of initial probes contained in the initial probe set to obtain an initial probe set capable of covering the entire target gene region, and for the final probe
  • further screening the initial probe set comprises: combining the DNA fragment (initial probe set) with the reference sequence The alignment is performed to obtain the number of alignments of each DNA fragment on the reference sequence, and the DNA fragments having more than one alignment are filtered out.
  • the initial probe set is further screened, in order to enable the final probe to capture the gene region in the same reaction system, and/or to cause the captured gene region to be eluted together under the same reaction conditions, including : Remove DNA fragments with a GC content other than 35-70%.
  • step (2) and step (3) comprise: (h) terminating the nucleic acid fragment to obtain a terminal repair fragment; (i) adding base A to the end repair fragment At both ends, a sticky end fragment is obtained; (j) a linker is ligated to both ends of the sticky end fragment to obtain a linker ligation fragment; (k) performing a first amplification of the linker ligation fragment to obtain a first amplification product; (1) capturing the first amplification product using the kit to obtain the target region; (m) performing second amplification on the target region to obtain a second amplification product; and (n) Right place The second amplification product is subjected to sequence determination to obtain the sequencing data.
  • the sequencing library construction included in steps (2) and (3) in the method of this aspect of the invention is particularly suitable for the construction of a sequencing library containing a trace amount of nucleic acid in a sample.
  • the sample contains a trace amount of free DNA.
  • the plasma sample contains an extremely small amount of target free DNA.
  • the first amplification allows the amount of nucleic acid to meet the needs of chip/probe hybridization capture.
  • the chip hybridization captures a certain amount of nucleic acid, and the second amplification enables capture.
  • the next target fragment is re-amplified to meet the requirements of on-machine sequencing and quality control detection.
  • This library construction method is particularly suitable for sequencing library construction of samples with a total free nucleic acid of not less than 10 ng or a conventional tissue genomic DNA of not less than 1 ⁇ g.
  • sequencing (2) and (3) in the method of this aspect of the invention includes sequencing of the constructed sequencing library
  • sequencing can be performed using known platforms including, but not limited to, Illumina's Hiseq2000/2500 platform, Life Technologies Ion Torrent platform and single molecule sequencing platform.
  • the sequencing method can be either single-ended sequencing or double-end sequencing. In one embodiment of the invention, double-end sequencing is utilized, and the resulting sequencing data consists of multiple pairs of read pairs.
  • the quality of the offline data after sequencing is high, and the high-quality offline data is beneficial for subsequent accurate detection and analysis. .
  • the step (4) comprises: performing a first alignment of the sequencing data with a reference sequence to obtain a first alignment result; and comparing the first alignment result with the reference sequence Part of performing a second alignment to obtain a second alignment result, wherein a part of the reference sequence includes each known InDel site in the target region reference sequence, and each of the known InDel sites is 1000 bp upstream and downstream a reference sequence; simultaneously detecting SNP, InDel, SV, and CNV variations in the target region based on the first alignment result and the second alignment result.
  • the second alignment is a local alignment
  • the first alignment is a conventional global alignment, and may be performed by using software such as SOAP or BWA according to its default setting to obtain a first comparison result, the first ratio.
  • the result includes the matching position of the read segment on the reference sequence and the matching situation information.
  • performing the second alignment that is, based on the first alignment result, locally re-aligning all the sequence information (reads) near all known INDELs in the reference sequence corresponding to the captured gene region, and eliminating the first ratio
  • the alignment error improves the accuracy of subsequent mutation detection, and the second alignment can be performed using the GATK comparison software ( https://www.broadinstitute.org/gatk/ ).
  • the SNP and INDEL variations are detected simultaneously by the GATK Unified Genotyper software.
  • the sequencing data is filtered before the first alignment, and the filtering includes removing the uncertainty base ratio by more than 10%.
  • the read range and/or the ratio of the number of bases whose base quality value is not more than 5 is not less than 50%.
  • the same pair of reads of the two of the read pairs of the first alignment result are removed.
  • a portion of the reference sequence includes each known InDel site in the target region reference sequence, and a reference sequence of 1000 bp each upstream and downstream of each known InDel site.
  • the reference sequence can be selected as a human reference genome, that is, a known human genome sequence, such as HG19, HG19 can be downloaded from NCBI, and the target region reference sequence is matched with the target region. That part of the reference genome sequence.
  • the step (4) further comprises: when at least one of the detected variations satisfies the following (i) or (ii), determining that the sample to be tested is a positive sample: (i) The number of reads supported in the negative control sample is less than 2 and the mutation rate in the positive control sample is greater than 1%, (ii) the sequencing depth is not less than 10X, and at least 3 reads are supported, in the negative control sample.
  • the number of reads supported by the reader was less than 2, the mutation rate in the positive control sample was greater than 1%, and the read support amount of the variant site was significantly different from the read support amount of the same site in the normal control sample.
  • the positive sample refers to the tumor sample.
  • the two determination conditions are determined by the inventor combined with the current relevant database information and a large amount of literature report information, a large number of positive samples and a large number of negative samples. It is statistically significant, and the latter is more than the former. Strictly, preferably, there are more than 30 positive or negative control samples.
  • the data of the control sample can be obtained by extracting and sequencing the nucleic acid of the control sample by itself, or by sequencing the samples in other publicly or publicly available databases. Data, multiple control sample data makes the statistical decision conditions/results statistically significant and more credible.
  • the results of the determination based on any two criteria can be used for clinical tumor diagnosis screening, and can be used to understand the type, possibility and development of cancer in the sample.
  • the read support amount of the variant site in the sample to be tested is significantly different from the read support amount of the same site of the normal control sample (negative control sample), wherein the read support amount can be
  • the number of reads in order to support the variation may also be the ratio of the reads supporting the variation in the read of the site in the alignment, in one embodiment of the invention, the latter is used for comparison, said Significant differences mean substantial differences, for example, for variant A in the sample to be tested, the ratio of reads in multiple positive samples (cancer samples) is 5/400 (variation 5 reads, total 400 reads), That is, the average mutation frequency of the site in the positive sample is 1.25%, and the ratio of reads support in the multiple negative control samples is 1/200 (variation 1 reads, total 200 reads), that is, the average in the negative control sample.
  • the frequency of variation is 0.5%. If the frequency of variation of the mutation site in the sample to be tested is closer to 1.25%, for example, 0.9%, the significant difference or the substantial difference is reached.
  • the difference between the read support ratio (variation frequency) of the variant site A and the mutation frequency of the site in the negative control sample for example, the z test or the t test can be used, and the difference is significant (p ⁇ 0.05), that is, Achieving significant differences.
  • the present invention further provides an apparatus for detecting a variation of a target area, which is used to implement or perform the target area variation detecting method of one or more embodiments of the present invention described above, the apparatus comprising: a data acquisition unit, configured to acquire sequencing data of the target region, where the sequencing data is composed of a plurality of read segments and/or a plurality of read segment pairs, and the data acquisition unit performs: acquiring nucleic acid in the sample to be tested
  • the nucleic acid is composed of a plurality of nucleic acid fragments derived from fragmented genomic DNA and/or free DNA, and the nucleic acid fragment is captured by a kit to obtain a target region, and the target region is subjected to sequence determination, wherein
  • the kit comprises a probe capable of specifically recognizing a predetermined region: a gene region of at least 10 of the 547 genes in Table 1;
  • a detecting unit configured to detect the target region variation based on the sequencing data from the data acquiring unit, the variation including at least
  • the detecting unit 200 in the device 1000 includes a first comparing subunit 13, a second comparing subunit 15, and a mutation identifying subunit 17,
  • the a comparison sub-unit 13 is configured to first compare the sequencing data from the data acquisition unit 100 with a reference sequence to obtain a first comparison result
  • the second comparison sub-unit 15 is configured to be from the first
  • the first alignment result of the comparison sub-unit 13 is second aligned with a portion of the reference sequence to obtain a second alignment result
  • the variation identification sub-unit 17 is configured to be based on the first alignment sub-unit a first alignment result of 13 and a second alignment result from the second alignment subunit 15 simultaneously detecting SNV, InDel, SV, and CNV variations in the target region to obtain mutation site information
  • a portion of the reference sequence includes each known InDel site in the target region reference sequence, and a reference sequence of 1000 bp each upstream and downstream of each known InDel site.
  • the detecting unit 200 of the apparatus 1000 further includes a first filtering subunit 12, the first filtering subunit 12 and the first comparing subunit 13 Connecting, for filtering the sequencing data before the sequencing data enters the first aligned sub-single 13 element, the filtering comprising removing a read having an undetermined base ratio of more than 10% and/or The ratio of the number of bases whose base quality value is not more than 5 is not less than 50%.
  • the filtering comprising removing a read having an undetermined base ratio of more than 10% and/or The ratio of the number of bases whose base quality value is not more than 5 is not less than 50%.
  • the detecting unit 200 further includes a second filtering subunit 14, the second filtering subunit 14 and the first comparing subunit 13 and the second alignment respectively
  • the subunit 15 is connected for removing one of the first alignment results from the first comparison subunit 13 before the first comparison result enters the second comparison subunit 15
  • the reference sequence may be HG19, the first alignment performed in the first comparison unit is a global alignment, and the second alignment performed in the second alignment sub-unit is a local alignment.
  • the detecting unit 200 in the apparatus 1000 further includes a determining subunit 19 for determining from the mutation identifying subunit 17. Whether the mutation site satisfies the following, and determines that the sample to be tested is a positive sample when at least one of the mutation sites satisfies the following: the number of read support in the negative control sample is less than 2 and in the positive control sample The mutation rate is greater than 1%.
  • the detecting unit 200 further includes a determining subunit 19 for determining whether the mutation site from the mutation identifying subunit 17 satisfies the following.
  • the at least one of the variant sites satisfies the following to determine that the sample to be tested is a positive sample: the sequencing depth is not less than 10X, the support of at least 3 reads, and the number of read support in the negative control sample is less than 2
  • the mutation rate in the positive control sample was greater than 1%, and the read support amount of the variant site was significantly different from the read support amount of the same site in the normal control sample.
  • a method of screening a tumor particularly an early screening, including but not limited to lung cancer, colorectal cancer, gastric cancer, breast cancer, renal cancer, pancreatic cancer, ovary Cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer, and liver cancer, the method comprising: obtaining nucleic acid in a sample to be tested, the nucleic acid consisting of a plurality of nucleic acid fragments derived from fragmented genomic DNA And/or free DNA; capturing the nucleic acid fragment using a kit to obtain a target region; performing sequence determination on the target region to obtain sequencing data, the sequencing data being composed of a plurality of reads; based on the sequencing data, detecting The variation in the target region, based on at least one of the detected mutations, satisfies the following (i) or (ii), and determines that the sample to be tested is a positive sample: (i) read support in the negative control sample The number of mutations
  • the predetermined area is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 of the 547 genes.
  • the combination of gene regions that can be specifically recognized by the kit probe in the method of the present invention is obtained by the inventor through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are common tumor occurrence or developmental correlations. region.
  • the common tumors include lung cancer, colorectal cancer, stomach cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer, and liver cancer.
  • the related gene sequences of a plurality of common cancers can be obtained in one time, simply and conveniently, and high specificity, and the related gene sequences can be detected and analyzed, and the detection analysis results can be used for early screening of various common cancers. Check and judge the possibility and effect of artificially intervening early tumor intervention. At present, most cancers such as lung cancer, liver cancer, and gastric cancer have been diagnosed at the time of hospital pathological diagnosis, which delays the earlier treatment time and greatly reduces the possibility of cure.
  • the predetermined region is a gene region of 145 genes listed in Table 2 of the 547 genes.
  • the gene region of the 145 genes in Table 2 that the probe can specifically recognize is obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are associated with the development of lung cancer.
  • all lung cancer-related gene sequences can be obtained in a single, simple, convenient and highly specific manner, and the information obtained by detecting these gene sequences can assist in early screening of lung cancer. diagnosis.
  • the predetermined region is a gene region of 60 genes listed in Table 3 of the 547 genes.
  • the gene regions of the 60 genes of Table 3 that the probe can specifically recognize are obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are associated with the development of colorectal cancer.
  • the probe in this kit in the method of the invention can acquire all colorectal cancer-related gene sequences in a single, simple, convenient and highly specific manner, and the information obtained by detecting these gene sequences can assist the early stage of colorectal cancer. Screening diagnosis.
  • the predetermined region is the gene region of the 43 genes listed in Table 4 of the 547 genes.
  • the gene regions of the 43 genes in Table 4 that the probe can specifically recognize are obtained by the inventors through multiple collections, multiple screenings, and multiple trial combinations. These gene region combinations are related to the occurrence and development of gynecological reproductive tract tumors.
  • the genital tract tumors include ovarian cancer, endometrial cancer, and cervical cancer.
  • the probe has a length of from 25 to 300 nt, preferably from 50 to 250 nt, and more preferably from 80 nt to 120 nt.
  • the probe is obtained by first obtaining an initial probe set and then screening the initial probe set. definite.
  • Obtaining the initial probe set includes: determining a reference sequence of the gene region, starting from one end of the reference sequence, sequentially acquiring a DNA fragment on the reference sequence to the other end of the reference sequence, wherein one DNA
  • the fragment is an initial probe
  • all of the DNA fragments constitute the initial probe set, the DNA fragments completely overlap, partially overlap or not overlap at all, and the initial probe set can cover the gene region at least once .
  • the reference sequence of the gene region can be obtained from the reference genome, for example, the corresponding gene region is obtained from the human reference genome HG19, and the corresponding gene region on all HG19 constitutes the reference sequence of the gene region, and HG19 can be NCBI database download.
  • the initial probe set is obtained by using an iterative algorithm design, including: determining a position of the gene region on a reference genome, and obtaining a reference sequence of the gene region, from the reference sequence
  • the first nucleotide begins to copy the reference sequence to obtain a first DNA fragment
  • the reference sequence is copied from the second nucleotide of the reference sequence to obtain a second DNA fragment, from which the reference sequence
  • the third nucleotide begins to copy the reference sequence to obtain a third DNA fragment, such that the subsequent DNA fragment is sequentially obtained until one end of the Nth DNA fragment exceeds the reference sequence, wherein one DNA fragment is an initial probe.
  • N is the total number of initial probes contained in the initial probe set to obtain an initial probe set capable of covering the entire target gene region, and for the final probe
  • further screening the initial probe set comprises: combining the DNA fragment (initial probe set) with the reference sequence The alignment is performed to obtain the number of alignments of each DNA fragment on the reference sequence, and the DNA fragments having more than one alignment are filtered out.
  • the initial probe set is further screened, in order to enable the final probe to capture the gene region in the same reaction system, and/or to cause the captured gene region to be eluted together under the same reaction conditions, including : Remove DNA fragments with a GC content other than 35-70%.
  • the kit captures the nucleic acid fragment, obtains a target region, and performs sequence determination on the target region to obtain sequencing data, including: (h) end-repairing the nucleic acid fragment, Obtain a terminal repair fragment; (i) adding base A to both ends of the end repair fragment to obtain a sticky end fragment; (j) attaching a linker to both ends of the sticky end fragment to obtain a linker fragment; (k) pair The linker ligation fragment is subjected to a first amplification to obtain a first amplification product; (1) capturing the first amplification product by the kit to obtain the target region; (m) targeting the target The region is subjected to a second amplification to obtain a second amplification product; and (n) the second amplification product is sequenced to obtain the sequencing data.
  • the sequencing library construction included in the method of this aspect of the invention is particularly suitable for the construction of a sequencing library containing a trace amount of nucleic acid in a sample.
  • the sample is a plasma sample containing a trace amount of free DNA, including extremely small targets. Free DNA, the first amplification enables the amount of nucleic acid to meet the needs of chip/probe hybridization capture, and the chip hybridization captures a certain amount of nucleic acid, and the second amplification enables the target fragment under capture to be re-amplified. Meet the requirements of sequencing and quality control testing.
  • This library construction method is particularly suitable for sequencing library construction of samples with a total free nucleic acid of not less than 10 ng or a conventional tissue genomic DNA of not less than 1 ⁇ g.
  • sequencing can be performed using known platforms including, but not limited to, Illumina's Hiseq2000/2500 platform, Life Technologies' Ion Torrent platform, and single molecule sequencing platform.
  • the sequencing method can be either single-ended sequencing or double-end sequencing. In one embodiment of the invention, double-end sequencing is utilized, and the resulting sequencing data consists of multiple pairs of read pairs.
  • the detecting the variation in the target area based on the sequencing data comprises: performing a first comparison with the reference sequence to obtain a first comparison result; Performing a second alignment with a portion of the reference sequence to obtain a second alignment result, the portion of the reference sequence including each known InDel site in the target region reference sequence, and A reference sequence of 1000 bp each upstream and downstream of each known InDel site is described; based on the first alignment result and the second alignment result, SNP, InDel, SV and CNV mutations in the target region are simultaneously detected.
  • the second alignment is a local alignment
  • the first alignment is a conventional global alignment, and may be performed by using software such as SOAP or BWA according to its default setting to obtain a first comparison result, the first ratio.
  • the result includes the matching position of the read segment on the reference sequence and the matching situation information.
  • performing the second alignment that is, based on the first alignment result, locally re-aligning all the sequence information (reads) near all known INDELs in the reference sequence corresponding to the captured gene region, and eliminating the first ratio
  • the alignment error improves the accuracy of subsequent mutation detection, and the second alignment can be performed using the GATK comparison software ( https://www.broadinstitute.org/gatk/ ).
  • the SNP and INDEL variations are detected simultaneously by the GATK Unified Genotyper software.
  • the mutation detecting method of the present invention it is possible to accurately detect a low frequency mutation having a mutation frequency of 1%.
  • the tumor screening method of this aspect of the invention the low frequency mutation with a mutation frequency of 1% can be accurately detected, which facilitates early screening of the tumor, assists in clinical diagnosis, and can prevent, treat, or monitor the tumor in time. .
  • the sequencing data is filtered before the first alignment, and the filtering includes removing the uncertainty base ratio by more than 10%.
  • Reading and / or alkali matrix A ratio of the number of bases having a magnitude of not more than 5 is not less than 50% of the read.
  • the same pair of reads of the two of the read pairs of the first alignment result are removed.
  • a portion of the reference sequence includes each known InDel site in the target region reference sequence, and a reference sequence of 1000 bp each upstream and downstream of each known InDel site.
  • the reference sequence can be selected as a human reference genome, that is, a known human genome sequence, such as HG19, HG19 can be downloaded from NCBI, and the target region reference sequence is matched with the target region. That part of the reference genome sequence.
  • the at least one of the detected variations satisfies the following (i) or (ii), and the sample to be tested is determined to be a positive sample, wherein the positive sample refers to a tumor
  • the sample may be a lung cancer, colorectal cancer, stomach cancer, breast cancer, kidney cancer, pancreatic cancer, ovarian cancer, endometrial cancer, thyroid cancer, cervical cancer, esophageal cancer or liver cancer samples.
  • the two judgment conditions are determined by the inventor in combination with the current relevant database information and a large amount of literature report information, a large number of positive samples and a large number of negative samples, which are statistically significant, and the latter is more strict than the former.
  • control sample there are more than 30 positive or negative control samples, and the data of the control sample can be obtained by extracting and sequencing the nucleic acid of the control sample by itself, or according to the sample sequencing data of the published or public database of others.
  • Multiple control sample data makes the statistical decision conditions/results statistically significant and more credible.
  • the results of the determination based on any two criteria can be used for clinical tumor diagnosis screening, and can be used to understand the type, possibility and development of cancer in the sample.
  • the read support amount of the variant site in the sample to be tested is significantly different from the read support amount of the same site of the normal control sample (negative control sample), wherein the read support amount can be
  • the number of reads in order to support the variation may also be the ratio of the reads supporting the variation in the read of the site in the alignment, in one embodiment of the invention, the latter is used for comparison, said Significant differences mean substantial differences, for example, for variant A in the sample to be tested, the ratio of reads in multiple positive samples (cancer samples) is 5/400 (variation 5 reads, total 400 reads), That is, the average mutation frequency of the site in the positive sample is 1.25%, and the ratio of reads support in the multiple negative control samples is 1/200 (variation 1 reads, total 200 reads), that is, the average in the negative control sample.
  • the frequency of variation is 0.5%. If the frequency of variation of the mutation site in the sample to be tested is closer to 1.25%, for example, 0.9%, the significant difference or the substantial difference is reached.
  • the difference between the read support ratio (variation frequency) of the variant site A and the mutation frequency of the site in the negative control sample for example, the z test or the t test can be used, and the difference is significant (p ⁇ 0.05), that is, Achieving significant differences.
  • the inventors have shown that the concentration of free plasma DNA (cfDNA) in normal human peripheral blood is 1-100 ng/mL, and the circulating tumor DNA (ctDNA) content in peripheral blood of tumor patients will increase significantly due to tumor cell secretion.
  • the genomic fragments produced by apoptosis or necrosis enter the blood, making the ctDNA content in the peripheral blood of tumor patients mean The degree can reach 180ng/mL, and the timing and monitoring of the changes and mutations of ctDNA in the peripheral blood of normal people and tumor patients can be applied to at least one of the following: early diagnosis of tumor, hereditary tumor prediction and state evaluation, Early detection of tumor progression, tumor detection and evaluation, tumor targeted therapy, chemotherapy for genetic variation analysis, tumor pathogenic gene trace residue detection, and tumor resistance gene mutation analysis.
  • minimally invasive the subject only needs to provide 5-10 mL of peripheral blood samples
  • Sex The subject can be tested for multiple times in real time. It can be detected regularly during early screening to monitor the risk of tumor. Tumor patients can be tested at any time after surgery, after chemotherapy/targeting, to analyze the prognosis of the operation and medication.
  • FIG. 1 is a schematic structural view of a target region variation detecting device in an embodiment of the present invention
  • FIG. 2 is a schematic structural diagram of a target region variation detecting device in an embodiment of the present invention.
  • FIG. 3 is a schematic structural diagram of a target region variation detecting device in an embodiment of the present invention.
  • Fig. 4 is a view showing the configuration of a target region variation detecting device in an embodiment of the present invention.
  • variants are common, and "SNP” (SNV), “CNV”, “insert deletion” (indel), and “structural variation” (SV) in the present invention are common.
  • SNP SNP
  • CNV CNV
  • insert deletion indel
  • SV structural variation
  • the definition is the same as usual, but the size of each variation is not particularly limited in the present invention, so that there are some crossovers between the several variations, such as when the insertion/deletion is a large fragment or even a whole chromosome, the copy number is also generated.
  • Mutation (CNV) or chromosomal aneuploidy also belongs to SV. The cross-over of the size of these types of variations does not prevent one skilled in the art from performing the methods and/or apparatus of the present invention and achieve the results described.
  • the "reference sequence” in the present invention is a known genomic sequence or at least a part of a known genomic sequence, and "first”, “second” and the like used in the present invention are merely for convenience of description and are not to be construed as indicating or Implying relative importance, it cannot be understood as having a sequential relationship.
  • the meaning of "multiple” is two or More than two.
  • a kit for obtaining an aspect of the invention, a method and/or apparatus for carrying out an aspect of the invention generally comprises designing a target region capture probe/chip, microsample construction and hybridization sequencing, and biological information analysis of the off-machine data. Interpretation of variant data.
  • an iterative algorithm is used to design a target region capture probe/chip that can be used or assisted for common cancer or specific cancer early screening diagnosis.
  • the target region includes driver genes related to common cancers or specific cancers, high-frequency mutation genes, important genes in 12 cancer-related signaling pathways, target drugs, and genes related to chemotherapy drugs.
  • the designed probe/chip can specifically identify the target region from a complex genome and capture the target region with high specificity and high coverage on the same set of probes/chips.
  • cfDNA plasma free DNA
  • ctDNA DNA derived from tumor cells
  • Linking Adapter library linker refers to a designed base sequence that binds to a primer when the cfDNA/ctDNA library is amplified, allows DNA amplification, and is sequenced with the primer when sequencing. The combination facilitates sequencing primer binding to the site to be sequenced for assisted DNA sequencing.
  • the library was subjected to the first round of PCR amplification
  • the library is quality-controlled after amplification and hybridized with the above probe/chip;
  • the hybrid library is subjected to a second round of PCR amplification
  • the Illumina HiSeq2500/2000 was sequenced on a machine with a sequencing depth of over 2000X.
  • SOAPnuke filter remove low quality reads
  • the individualized interpretation of the mutated data after bioinformatics analysis with reference to the constructed tumor database and related literature, analyzes the variation detected by the subject to assist in judging the tumor-related gene status of the tested sample, and whether there is cancer Risks, as well as the benign and malignant conditions of early tumor tissue, are used to assist in the most appropriate prevention and treatment methods in combination with clinical test results.
  • test results obtained using the method/device of the present invention are described in detail below in connection with specific individual samples.
  • the following examples are merely illustrative of the invention and are not to be construed as limiting the invention.
  • the reagents, sequences (linkers, tags and primers), software and instruments not specifically addressed in the following examples are conventional commercial products or open source, such as the hiseq2000 sequencing platform from Illumina. Library related kits for library construction and the like.
  • CANPer CANPer
  • CANPer is a liquid phase chip.
  • the CANPer chip includes the driver gene of the common high-risk cancer, the high-frequency mutation gene, and the important genes in the 12 signal pathways related to cancer, totaling 547 genes, 300Kb. The list of genes is shown in Table 1.
  • the peripheral blood plasma of patients with early stage of lung nodules was used as the sample to be tested.
  • the sample was from Tianjin Maternal and Child Health Hospital. The contents are as follows:
  • the separated plasma and remaining blood cells are stored in a -80 ° C refrigerator to avoid repeated freezing and thawing.
  • the filter column is empty at 8000 rpm for 1 min;
  • the filter column is 14000 rpm, 3 min;
  • the above-mentioned gene chip CANPer-1.75M entrusted by Roche was used, and hybridization capture and elution were carried out according to the instructions provided by the chip manufacturer. Finally, the magnetic beads were eluted by back-mixing with 21 ⁇ L of ddH 2 O.
  • the Illumina HiSeq2500 PE101+8+101 program was used for sequencing on the machine.
  • the sequencing experiment was performed according to the manufacturer's operating instructions (see Illumina/Solexa official cBot) for sequencing.
  • SOAPnuke filter removes n ⁇ 10% and the number of bases with a base mass value ⁇ 5 > 50% of reads;
  • Filt_bam remove the mismatch base ⁇ 3 reads
  • QC information such as the capture efficiency, effective number of reads, average depth, repetition rate, coverage, and uncovered intervals of the statistical chip;
  • the screening parameters used were: sequencing depth ⁇ 10X, variability in negative (normal) samples ⁇ 2%, variability in positive samples ⁇ 1%, and the number of reads supporting the variation in the sample data to be tested ⁇ 3, there is a significant difference between the read support ratio of the normal control (somatic cells) (p ⁇ 0.05);
  • chemotherapeutic drugs Annotate the function of variation, the number of reads support, the frequency of mutation, the variation of amino acids, and the variation in the database Cosmic; assist in determining the possible sources of disease based on the variation.
  • the killing effect of chemotherapeutic drugs on tumor cells is significantly correlated with the expression and/or polymorphism of a specific (a group of) genes.
  • the detection of related genes predicts the efficacy of chemotherapeutic drugs and selects appropriate drugs for individualized chemotherapy. It has become a reasonable choice to improve efficacy and reduce ineffective treatment.
  • the PharmGKB database is used to integrate all the current chemotherapeutic drugs and the genes related to curative effect and predictive evaluation of therapeutic effects, and to form a database for interpretation of individualized drugs for chemotherapy.
  • the chemotherapy data was integrated into the individualized information flow of the tumor to complete the automated interpretation of the chemotherapy drug.
  • Targeted drugs have the characteristics of significant drug efficacy and few side effects in tumor therapy, but they are dependent on targets (including protein, DNA, etc.). Target analysis must be performed on patients before they can determine whether patients can take drugs. Integrate current FDA-approved targeted drugs, as well as drugs in clinical III and IV. According to the NCCN clinical guidelines, the clinical drug gene research collates the relationship between the drug target gene and the target drug, and forms a database of individualized target drug interpretation.
  • the sample detected the 451 amino acid missense mutation of the EGFR gene, belonging to exon 12, which is located in the extracellular domain of the protein, which is not recorded in the COSMIC database, but the p.
  • the R451H] missense mutation was recorded once and reported to be associated with lung cancer (18948947). Functional predictions show that the mutation is a deleterious mutation that may have an impact on gene function.
  • the human epidermal growth factor receptor an expression product of the proto-oncogene c-erbB1 is a member of the receptor tyrosine kinase family.
  • EGFR is mainly located on the surface of the cell membrane, and activates its own tyrosine phosphorylation by binding to ligands. Autophosphorylation promotes downstream signaling pathways, including MPAK, PI3K and JNK pathways, and induces cell proliferation and differentiation. Mutations or abnormal expression of EGFR are present in many solid tumors.
  • the LungPer chip includes a lung cancer-related driver gene (Driver Gene), a high-frequency mutation gene, an important gene in 12 cancer-related signaling pathways, a target drug, and a chemotherapeutic drug-related gene, and a total of 145 genes, 250 Kb.
  • Driver Gene lung cancer-related driver gene
  • Table 2 The list of genes is shown in Table 2.
  • the sample detected the 451 amino acid missense mutation of the EGFR gene, belonging to exon 12, which is located in the extracellular domain of the protein, which is not recorded in the COSMIC database, but the p.
  • the R451H] missense mutation was recorded once and reported to be associated with lung cancer (18948947). Functional predictions show that the mutation is a deleterious mutation that may have an impact on gene function.
  • the human epidermal growth factor receptor an expression product of the proto-oncogene c-erbB1 is a member of the receptor tyrosine kinase family.
  • EGFR is mainly located on the surface of the cell membrane, and activates its own tyrosine phosphorylation by binding to ligands. Autophosphorylation promotes downstream signaling pathways, including MPAK, PI3K and JNK pathways, and induces cell proliferation and differentiation. Mutations or abnormal expression of EGFR are present in many solid tumors.
  • the ColorectalPer chip includes a driver gene related to colorectal cancer, a high-frequency mutated gene, an important gene in 12 cancer-related signaling pathways, a target drug, and a chemotherapeutic drug-related gene, and a total of 60 genes, as shown in Table 3. A total of 123Kb.
  • the samples are from Tianjin Maternal and Child Health Hospital.
  • This sample detected a missense mutation of KRAS p. [Gly12Asp], which was recorded 10,303 times in the COSMIC database, and about 60% were reported to be associated with the pathogenesis of colorectal cancer.
  • the KRAS codon 12 is located on the GTP domain and is the most common mutation in KRAS.
  • KRAS a member of the Ras gene family, encodes the P21 protein and functions in the MAPK signaling pathway. It is an oncogene that binds to GDP/GTP and promotes GTPase activity. When KRAS is mutated, it cannot be hydrolyzed by hydrolase, and it is in a state of continuous activation, which causes up-regulation of RAF/MAPK and transmits a variety of survival pathway signals, thereby allowing cells to overgrow and proliferate and resist EGFR-TKIs. Mutations can lead to a variety of malignancies, including lung cancer, mucinous adenomas, pancreatic ductal cancer, and colon cancer.
  • the most common way in which the KRAS gene is activated is point mutations, which occur at the N-terminal 12, 13, and 61, 146 codons, with the 12th codon mutation being the most common. Different mutation sites have different activation mechanisms for P21 protein.
  • the 12th codon mutation can attenuate the intrinsic GTPase activity of P21 and reduce apoptosis and decrease the inhibition of cell contact.
  • the encoded protein is one of the components of the DNA mismatch repair system (MMR), forming two different heterodimers: MutS ⁇ (MSH2-MSH6 heterodimer) and MutS ⁇ (MSH2-MSH3 heterodimer) Can be mismatched with DNA
  • MMR DNA mismatch repair system
  • MutS ⁇ or ⁇ forms a ternary complex with MutL ⁇ heterodimer, which is responsible for directing downstream MMR events, including strand recognition, excision, and resynthesis.
  • the binding and hydrolysis of ATP plays an important role in the mismatch repair function, and the ATPase activity is related to MutS ⁇ .
  • MutS ⁇ can also play a role in DNA homologous recombination repair function. This gene is associated with hereditary nonpolyposis colorectal cancer type I and endometrial cancer.
  • the WCNPer chip includes a driver gene related to gynecological genital tract tumors, a high-frequency mutated gene, an important gene in 12 signal pathways related to cancer, a target drug and a chemotherapeutic drug-related gene, and a total of 43 genes, as shown in Table 4. Show, a total of 300Kb.
  • the peripheral blood plasma of the subject was taken as the research object, and the sample was from Tianjin Maternal and Child Health Hospital. The experiment and data analysis were carried out with reference to Example 1.
  • This sample detected a missense mutation of BRAF p. [G469V], which was recorded 17 times in the COSMIC database and found in tumors such as lung, large intestine, biliary tract, upper respiratory tract, and esophagus.
  • the BRAF codon 469 is located in the ATP-binding domain of the protein kinase domain.
  • a melanoma study has shown that this mutation is an activating mutation that may cause BRAF to change from an inactive state to an active state or to abnormally activate the BRAF signaling pathway, and disease. The occurrence and development may be related.
  • the BRAF gene encodes a serine threonine protein kinase in the MAPK pathway, which transduces the signal from Ras to MEK1/2, thereby participating in the regulation of cell function and affecting cell sorting, differentiation and secretion. Mutations produced by this gene are associated with many types of cancer, such as colorectal cancer, lung cancer, liver cancer, pancreatic cancer, thyroid cancer, ovarian cancer, and the like. In ovarian cancer, the mutation frequency of BRAF gene is 8%, which is the driver gene in the development of ovarian cancer.
  • TP53 codon 266 is located in the sequence-specific DNA-binding domain and is an important domain for TP53 to function. This mutation may affect or lose the complete function of TP53.
  • TP53 is a driver gene in tumorigenesis and development, and its full function is affected. Impact or loss may be related to the development of the disease.
  • the TP53 gene is one of the genes most recently found to be associated with tumors. As an important tumor suppressor gene, it plays a key role in cell cycle regulation, DNA damage repair, cell differentiation, apoptosis and senescence. The TP53 gene is involved in more than 50% of human malignancies. Clinical studies have confirmed that 95.1% of p53 point mutations in tumors occur mainly at the highly conserved sites 175, 245, 248, 249, 273 and 282. Many tumor treatments are currently achieved by regulating TP53 protein. The TP53 gene has clinical application in a variety of cancers. Breast cancer patients with mutations in TP53 (exons 5-8) have a poor prognosis and tamoxifen has a significantly reduced efficacy. Mutation and loss of function of TP53 is one of the most common genetic abnormalities in ovarian cancer.
  • the subject detects important variability related to gynecological diseases, and combined with the clinical diagnosis, the risk and benign and malignant condition of the gynecological tumor can be judged.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne un procédé de détection de la variation d'une région cible, comprenant : l'obtention de l'acide nucléique d'un échantillon d'essai, l'acide nucléique étant composé d'une pluralité de fragments d'acides nucléiques ; la capture des fragments d'acides nucléiques à l'aide d'un kit pour obtenir la région cible ; le séquençage de la région cible pour obtenir les données de séquençage, les données de séquençage étant composées d'une pluralité de lectures ; et la détection de la variation de la région cible en fonction des données de séquençage. Le kit comprend les sondes capables de reconnaître spécifiquement la région prédéfinie suivante : région de gènes comportant au moins 10 des 547 gènes figurant dans le Tableau 1. Un dispositif pour détecter la variation de la région cible et une méthode de dépistage d'une tumeur sont en outre décrits.
PCT/CN2014/093871 2014-12-15 2014-12-15 Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible WO2016095093A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2014/093871 WO2016095093A1 (fr) 2014-12-15 2014-12-15 Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2014/093871 WO2016095093A1 (fr) 2014-12-15 2014-12-15 Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible

Publications (1)

Publication Number Publication Date
WO2016095093A1 true WO2016095093A1 (fr) 2016-06-23

Family

ID=56125560

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/093871 WO2016095093A1 (fr) 2014-12-15 2014-12-15 Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible

Country Status (1)

Country Link
WO (1) WO2016095093A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107723351A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种循环肿瘤dna肺癌驱动基因的高通量检测方法
CN107723352A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种循环肿瘤dna肝癌驱动基因高通量检测方法
CN107723364A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种结直肠癌易感基因的筛查方法
CN110570904A (zh) * 2019-08-27 2019-12-13 深圳百诺精准医疗科技有限公司 一种肿瘤突变分析方法、系统、终端及可读存储介质
CN110592208A (zh) * 2019-10-08 2019-12-20 北京诺禾致源科技股份有限公司 地中海贫血症三类亚型的捕获探针组合物及其应用方法和应用装置
CN110806479A (zh) * 2019-11-15 2020-02-18 复旦大学附属肿瘤医院 一种乳腺癌相关的激酶变异的检测panel及其应用
CN112086131A (zh) * 2020-08-18 2020-12-15 西安医学院 一种高通量测序中假阳性变异位点的筛选方法
CN112270952A (zh) * 2020-10-30 2021-01-26 广西师范大学 一种识别癌症驱动通路的方法
CN112739828A (zh) * 2018-06-11 2021-04-30 深圳华大生命科学研究院 确定待测样本类型的方法及系统
CN113355421A (zh) * 2021-07-03 2021-09-07 南京世和基因生物技术股份有限公司 肺癌早筛标志物、模型构建方法、检测装置以及计算机可读取介质
CN113373225A (zh) * 2021-06-10 2021-09-10 谱天（天津）生物科技有限公司 一种对临床样本基因和蛋白高通量检测结果的联合分析方法
CN113481299A (zh) * 2021-06-30 2021-10-08 苏州京脉生物科技有限公司 用于肺癌检测的靶向测序panel、试剂盒及获得靶向测序panel的方法
CN113736878A (zh) * 2021-08-24 2021-12-03 复旦大学附属肿瘤医院 一种神经系统肿瘤检测的基因panel、试剂盒及其应用
CN114410763A (zh) * 2022-02-11 2022-04-29 武汉艾迪康医学检验所有限公司 一种基于ngs的结直肠癌基因突变检测分析方法
CN115691665A (zh) * 2022-12-30 2023-02-03 北京求臻医学检验实验室有限公司 基于转录因子的癌症早期筛查诊断方法
CN115910349A (zh) * 2023-01-09 2023-04-04 北京求臻医学检验实验室有限公司 基于低深度wgs测序末端特征的癌症早期预测方法
CN116994656A (zh) * 2023-09-25 2023-11-03 北京求臻医学检验实验室有限公司 一种用于提高二代测序检测准确度的方法
WO2024138956A1 (fr) * 2022-12-30 2024-07-04 臻和(北京)生物科技有限公司 Procédé et appareil de détection de maladie résiduelle minimale, dispositif et support de stockage

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103119179A (zh) * 2010-07-23 2013-05-22 哈佛大学校长及研究员协会 用于检测体液中的疾病或病症标记的方法
WO2014039556A1 (fr) * 2012-09-04 2014-03-13 Guardant Health, Inc. Systèmes et procédés pour détecter des mutations rares et une variation de nombre de copies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103119179A (zh) * 2010-07-23 2013-05-22 哈佛大学校长及研究员协会 用于检测体液中的疾病或病症标记的方法
WO2014039556A1 (fr) * 2012-09-04 2014-03-13 Guardant Health, Inc. Systèmes et procédés pour détecter des mutations rares et une variation de nombre de copies

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107723351A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种循环肿瘤dna肺癌驱动基因的高通量检测方法
CN107723352A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种循环肿瘤dna肝癌驱动基因高通量检测方法
CN107723364A (zh) * 2016-08-12 2018-02-23 嘉兴允英医学检验有限公司 一种结直肠癌易感基因的筛查方法
CN112739828A (zh) * 2018-06-11 2021-04-30 深圳华大生命科学研究院 确定待测样本类型的方法及系统
CN112739828B (zh) * 2018-06-11 2024-04-09 深圳华大生命科学研究院 确定待测样本类型的方法及系统
CN110570904A (zh) * 2019-08-27 2019-12-13 深圳百诺精准医疗科技有限公司 一种肿瘤突变分析方法、系统、终端及可读存储介质
CN110592208B (zh) * 2019-10-08 2022-05-03 北京诺禾致源科技股份有限公司 地中海贫血症三类亚型的捕获探针组合物及其应用方法和应用装置
CN110592208A (zh) * 2019-10-08 2019-12-20 北京诺禾致源科技股份有限公司 地中海贫血症三类亚型的捕获探针组合物及其应用方法和应用装置
CN110806479A (zh) * 2019-11-15 2020-02-18 复旦大学附属肿瘤医院 一种乳腺癌相关的激酶变异的检测panel及其应用
CN112086131A (zh) * 2020-08-18 2020-12-15 西安医学院 一种高通量测序中假阳性变异位点的筛选方法
CN112086131B (zh) * 2020-08-18 2024-05-24 西安医学院 一种重测序数据库中假阳性变异位点的筛选方法
CN112270952B (zh) * 2020-10-30 2022-04-05 广西师范大学 一种识别癌症驱动通路的方法
CN112270952A (zh) * 2020-10-30 2021-01-26 广西师范大学 一种识别癌症驱动通路的方法
CN113373225A (zh) * 2021-06-10 2021-09-10 谱天（天津）生物科技有限公司 一种对临床样本基因和蛋白高通量检测结果的联合分析方法
CN113481299A (zh) * 2021-06-30 2021-10-08 苏州京脉生物科技有限公司 用于肺癌检测的靶向测序panel、试剂盒及获得靶向测序panel的方法
CN113481299B (zh) * 2021-06-30 2022-05-10 苏州京脉生物科技有限公司 用于肺癌检测的靶向测序panel、试剂盒及获得靶向测序panel的方法
CN113355421A (zh) * 2021-07-03 2021-09-07 南京世和基因生物技术股份有限公司 肺癌早筛标志物、模型构建方法、检测装置以及计算机可读取介质
CN113736878A (zh) * 2021-08-24 2021-12-03 复旦大学附属肿瘤医院 一种神经系统肿瘤检测的基因panel、试剂盒及其应用
CN114410763A (zh) * 2022-02-11 2022-04-29 武汉艾迪康医学检验所有限公司 一种基于ngs的结直肠癌基因突变检测分析方法
CN115691665A (zh) * 2022-12-30 2023-02-03 北京求臻医学检验实验室有限公司 基于转录因子的癌症早期筛查诊断方法
WO2024138956A1 (fr) * 2022-12-30 2024-07-04 臻和(北京)生物科技有限公司 Procédé et appareil de détection de maladie résiduelle minimale, dispositif et support de stockage
CN115910349A (zh) * 2023-01-09 2023-04-04 北京求臻医学检验实验室有限公司 基于低深度wgs测序末端特征的癌症早期预测方法
CN116994656A (zh) * 2023-09-25 2023-11-03 北京求臻医学检验实验室有限公司 一种用于提高二代测序检测准确度的方法
CN116994656B (zh) * 2023-09-25 2024-01-02 北京求臻医学检验实验室有限公司 一种用于提高二代测序检测准确度的方法

Similar Documents

Publication Publication Date Title
WO2016095093A1 (fr) Méthode de dépistage d'une tumeur, procédé et dispositif de détection d'une variation de la région cible
JP6700333B2 (ja) ヘテロ接合性の消失（ｌｏｓｓ ｏｆ ｈｅｔｅｒｏｚｙｇｏｓｉｔｙ）を評価するための方法および材料
CN106715723B (zh) 测定样品中pik3ca突变状态的方法
CN105779434A (zh) 试剂盒及其用途
TW201840853A (zh) 使用核酸片段之診斷應用
AU2016293025A1 (en) System and methodology for the analysis of genomic data obtained from a subject
CN113151474A (zh) 用于癌症检测的血浆dna突变分析
CN105780129B (zh) 目标区域测序文库构建方法
Ledgerwood et al. The degree of intratumor mutational heterogeneity varies by primary tumor sub-site
CN107849569B (zh) 肺腺癌生物标记物及其应用
EP3638815A1 (fr) Procédé pour déterminer la sensibilité d'un patient atteint d'une maladie proliférative à un traitement utilisant un agent qui cible un composant de la voie pd1/pd-l1
CN105779435A (zh) 试剂盒及其用途
CN104032001B (zh) 用于胆囊癌预后评估的erbb信号通路突变靶向测序方法
Puls et al. Molecular pathology of bone tumours: diagnostic implications
CN105779432A (zh) 试剂盒及其用途
Hirsch et al. Molecular characterization of ulcerative colitis-associated colorectal carcinomas
US20230002831A1 (en) Methods and compositions for analyses of cancer
CN110004229A (zh) 多基因作为egfr单克隆抗体类药物耐药标志物的应用
JP5865241B2 (ja) 肉腫の予後分子署名およびその使用
US20220298565A1 (en) Method Of Determining PIK3CA Mutational Status In A Sample
CN107974504A (zh) 基于ngs方法的肺癌和结直肠癌基因检测的方法
US20180044722A1 (en) Tri-color probes for detecting multiple gene rearrangements in a fish assay
CA3151627A1 (fr) Utilisation d'une detection simultanee de marqueurs pour evaluer le gliome diffus et la reactivite a un traitement
CN108342488A (zh) 一种用于检测胃癌的试剂盒
US9976184B2 (en) Mutations in pancreatic neoplasms

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14908128

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14908128

Country of ref document: EP

Kind code of ref document: A1