CN108588201A - A kind of method and device of colorectal cancer Cetuximab drug resistance trace amount DNA abrupt climatic change - Google Patents
A kind of method and device of colorectal cancer Cetuximab drug resistance trace amount DNA abrupt climatic change Download PDFInfo
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Abstract
The invention discloses a kind of method and device of colorectal cancer Cetuximab drug resistance trace amount DNA abrupt climatic change, the method includes：Dissociative DNA is extracted from sample；The dissociative DNA is built into library；The library is enriched with；The library of the enrichment is captured using capture probe, obtains capture dna；The capture dna is sequenced, sequencing result is obtained；By the sequencing result compared with reference sequences, mutational site is detected.The method of the invention realizes low initial amount DNA to be enriched with capture sequencing abrupt climatic change.
Description
Technical field
The invention belongs to biotechnology, especially cancer genes to detect, and particularly relate to a kind of knot
The method and device of carcinoma of the rectum Cetuximab drug resistance trace amount DNA enrichment capture sequencing abrupt climatic change.
Background technology
Colorectal cancer is most common tumor in digestive tract in world wide.It is counted according to authoritative institution, the annual neopathy in the whole world
Example is more than Isosorbide-5-Nitrae 00,000, and death is close to 700,000, before either the equal shelter of incidence or the death rate has malignant tumour
It arranges [1].In the U.S., with constantly universal and perioperative treatment mode the constantly improve of early screening, limitation and area
5 years survival rates of domain property colorectal cancer are up to 70% or more, but 5 years survival rates of metastatic colorectal carcinoma are still 13%-14%
Left and right, prompts the prevention and control situation still very severe [2] of colorectal cancer.Cetuximab (Cetuximab) is species specificity resistance
The IgG monoclonal antibody of disconnected EGF-R ELISA EGFR (epidermal growth factor receptor), it is multinomial
Clinical research shows that in a line or second line treatment, it is straight that chemotherapy regimen joint Cetuximab can significantly improve metastatic knot
The survival region [3-5] of patients with bowel cancer.
It is mostly at present that RAS families, BRAF are carried out based on tissue sample for the detection method of Cetuximab outcome prediction
The PCR or generation Sanger sequencing approaches of gene.These methods have limitation below：(1) relapse and metastasis corrective surgery
The testing result of tissue detection is inaccurate.For relapse and metastasis colorectal cancer patients, sample of tissue when what is mostly used is operation
Carry out Molecular Detection.Temporal heterogeneity caused by tumour evolution can make sample segment detection that false negative result be presented.Simultaneously
The genome that inside tumor and different parts transfer kitchen all have space aspects is heterogeneous, and the sampling of biopsy is organized to deviate
It can influence the accuracy of testing result.(2) transfer of cancer cell may be caused when tissue biopsy, and there is certain clinical risk；
(3) detection gene range is small.(4) sensitivity of quantitative fluorescent PCR or Sanger detection platforms is limited, cannot accurately detect low
The gene mutation of abundance.
It is extracted in cast-off cells, aspiration biopsy trace sample, peripheral blood dissociative DNA sample how to efficiently use coming from
Trace amount DNA carries out genetic test, is to solve the problems, such as colorectal cancer Cetuximab Drug Resistance Detection problem.
Bibliography
1.Torre LA,Bray F,Siegel RL et al.Global cancer statistics,2012.CA
Cancer J Clin.2015Mar；65(2):87-108.doi:10.3322/caac.21262.
2.Siegel RL,Miller KD,Fedewa SA et al.Colorectal cancer statistics,
2017.CACancer J Clin.2017May 6；67(3):177-193.doi:10.3322/caac.21395.
3.Sobrero AF,Maurel J,Fehrenbacher L et al.EPIC:phase III trial of
cetuximab plus irinotecan after fluoropyrimidine and oxaliplatin failure in
patients with metastatic colorectal cancer.J Clin Oncol.2008May 10；26(14):
2311-9.doi:10.1200/JCO.2007.13.1193.
4.Venook AP,Niedzwiecki D,Lenz HJ et al.Effect of First-Line
Chemotherapy Combined With Cetuximab or Bevacizumab on Overall Survival in
Patients With KRAS Wild-Type Advanced or Metastatic Colorectal Cancer:A
Randomized Clinical Trial.JAMA.2017Jun 20；317(23):2392-2401.doi:10.1001/
jama.2017.7105.
5.Segelov E,Waring P,Desai J et al.ICECREAM:randomised phase II study
of cetuximab alone or in combination with irinotecan in patients with
metastatic colorectal cancer with either KRAS,NRAS,BRAF and PI3KCA wild type,
or G13D mutated tumours.BMC Cancer.2016May 31；16:339.doi:10.1186/s12885-016-
2389-8.
Invention content
The present invention is carried in view of the characteristic for being not easy to obtain of colorectal cancer patients tissue samples DNA or peripheral blood dissociative DNA
A kind of method of low initial amount DNA enrichments capture sequencing abrupt climatic change is supplied, to overcome the shortcomings of original technology.
Therefore, in a first aspect, the present invention provides a kind of mutation of colorectal cancer Cetuximab drug resistance trace amount DNA to examine
The method of survey, the method includes the steps：
(1) DNA is extracted respectively from examined samples and check sample；
(2) DNA is built into library respectively；
(3) library is enriched with respectively；
(4) library of the enrichment is captured respectively using capture probe, obtains capture dna；
(5) capture dna is sequenced, obtains sequencing result；
(6) by the sequencing result of the examined samples and the check sample, mutational site is detected.
In one embodiment, the method further includes (7) for detected mutational site, calculates the mutational site and exists
The frequency of mutation in library.
In one embodiment, the dissociative DNA is divided into two parts in step (2) while being built two libraries,
For detected mutational site in step (7), the mutational site average frequency of mutation in two libraries is calculated, as the mutation position
The frequency of mutation after the correction of point.
In one embodiment, in step (6), for multiple samples, mutational site detection is as follows：
Obtain the vision response test μ of the capture probe of each sample and the locus specificity error rate e of multiple samples；
It calculates in examined samples, sequencing read number occurs for mistake expected from mutated site, which is divided into whole mistake
Accidentally sequencing read desired value NμWith the wrong desired value N of locus specificitye, formula is as follows：
Nμ=D* μ
Ne=D*e
Wherein, total sequencing depth of D mutated sites in examined samples,
It assesses in examined samples respectively, the mutation sequencing actually detected several N of read occur that read number desired value is sequenced with mistake
Confidence level p in Poisson distributionNμAnd pNe, formula is as follows：
pNμ=1-ppois (N, Nμ)
pNe=1-ppois (N, Ne)
If pNμAnd pNeIt is less than conspicuousness threshold value, and check sample mutation sequencing read number≤2, the i.e. point mutation simultaneously
Really to detect mutation.
In one embodiment, the conspicuousness threshold value 0.05, more optional 0.01.
In one embodiment, the vision response test μ of the capture probe is calculated：Formula is as follows,
Wherein n is the base number of capture probe, and i is some site in capture probe, MiFor the false bases number in the sites i, Di
For the total depth in the sites i.
In one embodiment, locus specificity error rate e is calculated：Formula is as follows,
Wherein, m is the total number of samples of detection, and j is the sample for specific site occurring, base mutation (such as G sports T) occurring,
MjFor in j samples, the sequencing read of the base mutation type supports number, DjIt is deep for total sequencing of the mutated site in j samples
Degree.
In one embodiment, in step (6), with GATK it is soft carry out somatic InDel variations (body cell be inserted into/
Deletion mutation) detection；CNV (genome copies number variation) detections are carried out with CONTRA softwares；Parameter is acquiescence.
In one embodiment, in step (1), the sample be selected from aspiration biopsy sample, saliva, pleural effusions and ascites,
Urine, excrement etc..
In one embodiment, PCR is carried out in step (3) be enriched with the library.The PCR primer, it is preferred to use
High-flux sequence universal primer.
In one embodiment, the gene order of listed gene during capture probe is for table 1 in step (4)
Probe.
In one embodiment, capture probe is for listed gene mutation in table 2 or table 3 in step (4)
Probe.
In second aspect, the present invention provides a kind of colorectal cancer Cetuximab drug resistance trace amount DNA abrupt climatic changes
Device, described device include：
DNA sample extraction module, for extracting DNA respectively from examined samples and check sample；
Library module is built, for the DNA to be built library respectively；
Library enrichment module, for the library to be enriched with respectively；
Trapping module obtains capture dna for capturing the library of the enrichment using capture probe；
Sequencer module obtains sequencing result for the capture dna to be sequenced；
It is mutated check-out module, according to the sequencing result of the examined samples and the check sample, detects mutational site.
In one embodiment, described device further includes：Frequency of mutation computing module, for detected mutational site,
Calculate the frequency of mutation in the mutational site.
In one embodiment, it is carried out in building library module：The dissociative DNA is divided into two parts while building two texts
Library carries out in frequency of mutation computing module, and for detected mutational site, it is averagely prominent in two libraries to calculate the mutational site
Frequency, as the frequency of mutation after the correction in the mutational site.
In one embodiment, mutational site detection is carried out in being mutated check-out module：
Obtain the vision response test μ of the capture probe of each sample and the locus specificity error rate e of multiple samples；
It calculates in examined samples θ, sequencing read number occurs for mistake expected from mutated site, which is divided into whole mistake
Accidentally sequencing read desired value NμWith the wrong desired value N of locus specificitye, formula is as follows：
Nμ=D* μ
Ne=D*e
Wherein, total sequencing depth of D mutated sites in sample θ,
Respectively in assessment examined samples θ, the actually detected several N of mutation sequencing readθSequencing read number occurs with mistake it is expected
It is worth the confidence level p in Poisson distributionNμAnd pNe, formula is as follows：
pNμ=1-ppois (Nθ,Nμ)
pNe=1-ppois (Nθ,Ne)
If pNμAnd pNeIt is less than conspicuousness threshold value, and check sample mutation sequencing read number≤2, the i.e. point mutation simultaneously
Really to detect mutation.
In one embodiment, the conspicuousness threshold value 0.05, more optional 0.01.
In one embodiment, the vision response test μ of the capture probe is calculated in being mutated check-out module：Formula is such as
Under,
Wherein n is the base number of capture probe, and i is some site in capture probe, MiFor the false bases number in the sites i, Di
For the total depth in the sites i.
In one embodiment, locus specificity error rate e is calculated in being mutated check-out module：Formula is as follows,
Wherein, m is the total number of samples of detection, and j is the sample for specific site occurring, base mutation (such as G sports T) occurring,
MjFor in j samples, the sequencing read of the base mutation type supports number, DjIt is deep for total sequencing of the mutated site in j samples
Degree.
In one embodiment, in being mutated check-out module, with GATK is soft somatic InDel variations is carried out (body is thin
Born of the same parents' insertion/deletion makes a variation) detection；CNV (genome copies number variation) detections are carried out with CONTRA softwares；Parameter is acquiescence.
In one embodiment, in DNA sample extraction module, the sample is selected from aspiration biopsy sample, saliva, chest
Seroperitoneum, urine, excrement etc..
In one embodiment, PCR is carried out in the enrichment module of library be enriched with the library.The PCR primer, preferably
Using high-flux sequence universal primer.
In one embodiment, capture probe is the gene order for listed gene in table 1 in trapping module
Probe.
In one embodiment, capture probe is for listed gene mutation in table 2 or table 3 in trapping module
Probe.
In the third aspect, the present invention provides a kind of capture chip, the capture chip includes for listed base in table 1
The capture probe of the gene order of cause.
In one embodiment, capture probe is for listed gene mutation in table 2 or table 3 in step (4)
Probe.
In fourth aspect the present invention provides the gene order combination for being used to indicate cancer of pancreas, the assortment of genes includes table
The gene order of listed gene in 1.
In one embodiment, the gene order combination includes gene mutation site listed in table 2 or table 3.
Relative to other analysis methods, the advantage of method of the invention is as follows：
1) high applicability：Relative to traditional Sanger sequencing approaches, this method only need down to 10ng DNA sample i.e.
Genetic test can be carried out, applicability of the molecular detecting method in cancer of pancreas sample is increased；
2) high-throughput：In conjunction with the experimental method of next-generation sequencing technologies and hybrid capture, can simultaneously to multiple samples into
Row detection, and colorectal cancer Cetuximab drug resistance related gene can be disposably detected, more fully mutation letter is provided
Breath；
3) high flexibility：Compared to traditional Molecular Detection means, this method may be implemented to single patient, segmentum intercalaris when more
Point sample or multizone sample, are continuously detected so that detection is more flexible；
4) high accuracy：By the sequencing of high depth, the sensibility of detection mutation can be improved, realizes that abundance is 0.1%
The accurate detection of mutation.
Description of the drawings
By the following drawings, the present invention will be described：
Fig. 1 shows colorectal cancer peripheral blood dissociative DNA enrichment capture sequencing schematic diagram.
Specific implementation mode
The pretherapy and post-treatment peripheral blood of colorectal cancer patients Cetuximab is efficiently utilized the purpose of the present invention is to provide a kind of
The method that sample carries out genetic test, with accurate evaluation Cetuximab drug resistance state and resistance mechanism.This method provides micro-
The preparation method of DNA (nanogram level) sample is measured, and provides the library constructing method of optimization and the enrichment chip pair of autonomous Design
DNA library carries out efficient capture, further the method for high-flux sequence is used to be detected (Fig. 1) to polygenes region, can be with
Efficiently, capture region gene mutation state, the primary to Cetuximab or secondary resistance reason are accurately, flexibly assessed
It is assessed, and further the therapeutic strategy of patient is optimized.
In the present invention, Gene Name is all made of NCBI-Gene (https://www.ncbi.nlm.nih.gov) it is inner
Official names (Official Symbol).The reference sequences of chromosome sequence are GRCh37/hg19 (http://
Hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/), the chromosome involved in the present invention is compiled
Number and position be also based on reference sequences be GRCh37/hg19.
In the present invention, the method for detection gene mutation site is not related to the diagnosis of disease merely, but dashes forward with certain genes
The method for becoming the detection gene mutation that site is combined can be used for disease detection.Therefore, the method for the present invention includes non-diagnostic
Method.
In the present invention, examined samples and check sample are preferred from the different samples of same subject.For example, test sample
This is blood plasma, and check sample is the haemocyte after peripheral blood separation.After the peripheral blood separation of subject, the blood plasma that is matched and
Haemocyte carries out building library.Every part of blood plasma is built into two libraries (DNA divides equally), every part of haemocyte builds a library.Blood plasma library is
Subject's as a result, the corresponding haemocyte result respectively matched.
Embodiment
1. capture probe designs：
Experience accumulation in being worked according to inventor, the relevant gene of colorectal cancer Cetuximab drug resistance that will build up on become
Different information carries out confluence analysis, carries out capture chip design, will be complete with the relevant 11 hot spot genes of Cetuximab drug resistance
Portion's exon region is designed into capture chip, such as following table.
Table 1：Western appropriate former times drug resistance associated hot point gene list
| KRAS | NRAS | ERBB2 | MET | EGFR |
| BRAF | PIK3CA | PTEN | MEK1 | NF1 |
| AKT1 |
2. peripheral blood dissociative DNA extracting method
2.1 peripheral blood acquisition methods
Using Streck blood sampling blood sampling tubes, blood sampling volume is not less than 15ml, and turn upside down mixing immediately after blood sampling, complete in 72h
It is detached at blood plasma.
2.2 blood plasma separation process
The isolated plasma sample of peripheral blood and blood cell sample, plasma sample and blood cell sample constitute pairing sample
Product.It is appreciated that in the present invention, check sample.Contain a small amount of gene mutated in colorectal carcinoma, and haemocyte in plasma sample
Tumor mutant gene is free of in sample, and for the rejecting of genetic background mutation, a small amount of knot is distinguished from a large amount of genetic backgrounds
Carcinoma of the rectum mutator, which is the present embodiment, to be solved the problems, such as.
(1) by peripheral blood, 1600g centrifuges 10min under the conditions of 4 DEG C, and upper plasma is dispensed into multiple 1.5mL after centrifugation
Or in the centrifuge tube of 2.0mL, pay attention to that intermediate leukocytic cream cannot be drawn onto during drawing blood plasma；Lower layer's lymphocyte and blood
Sample is preserved cell as a contrast.
(2) plasma sample dispensed is subjected to secondary separation, 16000g centrifuges 10min under the conditions of 4 DEG C, and supernatant is turned
It moves on in the centrifuge tube of new 1.5mL or 2.0mL, discards residual white；
Blood plasma and check sample label are clear after detaching twice, and it is spare to be stored in -80 DEG C of refrigerators.
2.3 plasma DNAs and check sample DNA extractions
Plasma DNA is extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN) dissociative DNA
Kit is completed.
The extraction of check sample DNA is carried out according to SDS cracking process.
The present invention can use the sample type of other extractable dissociative DNAs, such as saliva, pleural effusions and ascites, urine, excrement
Deng；The peripheral blood acquisition that other heparin tubes carry out；Other methods collect pancreatic juice and biopsy sample；The DNA that other kits carry out
Extraction.
3. library construction：
In view of normal DNA abundance is high in peripheral blood, mutant DNA abundance is extremely low, to reduce normal DNA background, improves low
The recall rate of frequency mutation, therefore use the strategy of double library constructions.Specifically, be two parts by the dissociative DNA average out to after extraction, and
The parallel structure in double libraries is carried out in the follow-up process.
Check sample DNA carries out building library.
Library reaction is built, library specification is built according to KAPA LTP Library Preparation Kit, it is anti-to carry out 3 step enzymatics
It answers.Double library constructions can effectively reduce the complexity of single library DNA molecule.
It repairs 3.1 ends
Later, 120 μ L of Agencourt AMPure XP reagent are added, carry out magnetic beads for purifying.Purified product band magnetic
Pearl carries out back dissolving, and back dissolving solvent is ddH2O, 42 μ L of back dissolving system, and next step reaction is carried out with magnetic bead, caused by avoiding tube
Sample loss.
3.2 plus A reactions
90 μ L of PEG/NaCl SPRI solution are added later, are sufficiently mixed, carry out magnetic beads for purifying, last back dissolving (35- connectors)
μL ddH2O, band magnetic bead carry out next step reaction.
3.3 connectors (Adapter) connect
It has used quick coupled reaction system, has substantially reduced the reaction time (16h shortens to 15min).
It is separately added into 50 μ L of PEG/NaCl SPRI solution later 2 times, carries out 2 magnetic beads for purifying, 25 μ L of last back dissolving
ddH2O。
The enrichment of 3.4 linking libraries
Enrichment PCR is carried out to library using the enrichment method of optimization, amplification enzyme selects Phusion HotStart
Polymerase, and DMSO is added to eliminate influence of the DNA secondary structures to amplification, specific reaction system is as follows.
Using fluorescence quantitative PCR instrument (ABI 7500), condition setting is such as
Agencourt AMPure XP reagent 50uL are added later and carry out magnetic beads for purifying.
Embodiment
Below by the retrospective inspection of peripheral blood dissociative DNA of the metastatic colorectal cancer patients of specific 50 postoperative recurrences
Acetonideexample is surveyed, the present invention will be described.50 patients to the past surgical tissue carried out RAS genes (KRAS's
The codon 12/13/61 of 12/13/61/146 codon, NRAS) and BRAF V600E a generation sequencing, be as a result shown as detecting
Site wild type, but in the therapeutic process for receiving joint Cetuximab, show as initial drug-resistant.In addition just with 100
The peripheral blood dissociative DNA of ordinary person compares.Peripheral blood and the preceding biopsy sample for the treatment of have carried out retrospective before being treated to patient
High throughput capture sequencing.High-throughput capture sequencing has been carried out to the peripheral blood sample of normal person.It should be noted that the embodiment
It is merely to illustrate that purpose, and limitation of the present invention cannot be construed in any way.
1. peripheral blood dissociative DNA extracts
1.1 peripheral bloods acquire
Using Streck blood sampling blood sampling tubes, blood sampling volume is not less than 15ml, and turn upside down mixing immediately after blood sampling, complete in 72h
It is detached at blood plasma.
1.2 blood plasma separation process
(1) by peripheral blood, 1600g centrifuges 10min under the conditions of 4 DEG C, and upper plasma is dispensed into multiple 1.5mL after centrifugation
Or in the centrifuge tube of 2.0mL, pay attention to that intermediate leukocytic cream cannot be drawn onto during drawing blood plasma；Lower layer's lymphocyte and blood
Sample is preserved cell as a contrast.
(2) plasma sample dispensed is subjected to secondary separation, 16000g centrifuges 10min under the conditions of 4 DEG C, and supernatant is turned
It moves on in the centrifuge tube of new 1.5mL or 2.0mL, discards residual white；
Blood plasma and check sample label are clear after detaching twice, and it is spare to be stored in -80 DEG C of refrigerators.
1.3 plasma DNAs and check sample DNA extractions
Plasma DNA is extracted using QIAamp Circulating Nucleic Acid Kit (QIAGEN) dissociative DNA
Kit is completed.
The extraction of check sample DNA is carried out according to SDS cracking process.
2. library construction：
Dissociative DNA after extraction is divided into the parallel structure for carrying out two libraries, to reduce DNA complexities in library, improves
Low frequency is mutated recall rate.Check sample DNA carries out building library.Library reaction is built, according to KAPA LTP Library Preparation
Kit builds library specification, carries out 3 step enzymatic reactions.
It repairs 2.1 ends
Later, 120 μ L of Agencourt AMPure XP reagent are added, carry out magnetic beads for purifying.Purified product band magnetic
Pearl carries out back dissolving, and back dissolving solvent is ddH2O, 42 μ L of back dissolving system, and next step reaction is carried out with magnetic bead, avoid removal magnetic bead mistake
Since DNA profiling caused by tube loses in journey.
2.2 plus A reactions
90 μ L of PEG/NaCl SPRI solution are added later, are sufficiently mixed, carry out magnetic beads for purifying, last back dissolving (35- connectors)
μL ddH2O, band magnetic bead carry out next step reaction.
2.3 connectors (Adapter) connect
It has used quick coupled reaction system, has substantially reduced the reaction time (16h shortens to 15min).Add respectively later
Enter 50 μ L of PEG/NaCl SPRI solution 2 times, carries out 2 magnetic beads for purifying, 25 μ L ddH of last back dissolving2O
The enrichment of 2.4 linking libraries
Enrichment PCR is carried out to library using the enrichment method of optimization, amplification enzyme selects Phusion HotStart
Polymerase, and DMSO is added to eliminate influence of the DNA secondary structures to amplification, specific reaction system is as follows.
Using fluorescence quantitative PCR instrument (ABI 7500), condition setting is such as
Agencourt AMPure XP reagent 50uL are added later and carry out magnetic beads for purifying.
It is sequenced with upper machine 3. probe is captured
3.1 high-throughput captures sequencings are to carry out probe covering per~100bp according to reference sequences according to list of genes,
Designed successively from front to back every the probe that 10bp designs a 100bp in the sections certain gene such as 1~3kb.After amplification
After library Quality Control qualification and using inventor design capture probe, with reference to chip manufacturer (Roche) provide specification into
Row hybrid capture.Finally 21 μ L ddH of elution back dissolving2O band hybridization elution magnetic beads.
The amplification of 3.2 hybrid capture products
PCR reaction systems：
PCR reaction conditions：
3.3 first remove previous step magnetic bead, then rejoin 50 μ L of Agencourt AMPure XP reagent, carry out
Magnetic beads for purifying, 25 μ L ddH2O of last back dissolving carry out QC and upper machine
3.4 carry out machine sequencing using Illumina HiSeq3000PE151 programs, and sequencing experimental implementation is according to manufacturer
The operational manual (announcing cBot referring to Illumina/Solexa officials) of offer carries out upper machine sequencing procedures.
4. data processing and mutation detection
4.1 times machine data are filtered, and reject low-quality sequencing read；
4.2 times machine data are compared using BWA softwares to reference gene group；
4.3 compare the label that bam files carry out repetitive sequence using Picard；
Bam files after 4.4 deduplications carry out the place of IndelRealigner and BaseRecalibrator using GATK
Reason.
4.5 single library mutation detections：
Abrupt climatic change process：Or base variation is random error or is really to be mutated, the H0 of this statistic processes assumes
Be be sequenced base variation be random error, then falsfication is not random mistake if p value and H0 are significant differences
Accidentally, as true mutation.Poisson distribution verification is carried out to the mutation detection in single library.Calculate separately detection mutation with two kinds with
The significance of difference of machine mistake (chip random sequencing mistake, sample random error).If mutation sequencing read supports number and two kinds
The significant difference of mistake, that is, think that this sports true mutation rather than random error.
4.5.1 each position of capture region is likely to that mistake occurs, so calculating the average error of whole region
Rate, i.e., the error rate adduction of each position, then divided by the size of whole region.
For each plasma sample, the vision response test of capture probe is calculated；In this sample, each chip site has
Certain error rate calculates average value.
Calculate the vision response test μ in capture section：Formula is as follows,
Wherein n number of sites between trapping region, i are some site in section, MiFor the false bases number in the sites i, DiIt is i
The total depth of point.
For all serum samples, the vision response test μ for capturing section is averaged.
4.5.2 for all serum samples, locus specificity error rate e in group is calculated：Formula is as follows,
Wherein, m is the total number of samples of detection, and j is the sample for specific site occurring, base mutation (such as G sports T) occurring,
MjFor in j samples, the sequencing read of the base mutation type supports number, DjIt is deep for total sequencing of the mutated site in j samples
Degree.
What above two step calculated is the incidence of random error.
4.5.3 it calculates if it is random error, under the sequencing depth of this part of blood plasma, it may appear that how many wrong sequencings
Read reads.It is divided into two parts, the sequencing read desired value of chip random error --- i.e. if it is chip random error, meeting
How many reads；The sequencing read of site random error --- i.e. if it is site random error, how many reads understood.
Two parts carry out significance test respectively.
It calculates in examined samples θ, sequencing read number occurs for mistake expected from mutated site, which is divided into whole mistake
Accidentally sequencing read desired value NμWith the wrong desired value N of locus specificitye, formula is as follows：
Nμ=Dθ*μ
Ne=Dθ*e
4.5.4 significance test calculates the notable of reads and two desired distribution actually occurred according to Poisson distribution
Property.
Respectively in assessment examined samples θ, the actually detected several N of mutation sequencing readθSequencing read number occurs with mistake it is expected
It is worth the confidence level p in Poisson distributionNμAnd pNe, formula is as follows：
pNμ=1-ppois (Nθ,Nμ)
pNe=1-ppois (Nθ,Ne)
If pNμAnd pNeSimultaneously less than 0.05, i.e., mutation sequencing read really detects number with sequencing random error with aobvious
Write difference, then it is assumed that the mutation is not caused by random error.
Above two step completes significance test.
It removes except conspicuousness, then is compared with check sample (result of haemocyte sequencing), if check sample simultaneously
Detection sequencing read number>2, that is, it is not considered as that the mutation is individual cells mutation.
4.5.5 somatic InDel variations (variation of body cell insertion/deletion) detection is carried out with GATK is soft；Use CONTRA
Software carries out CNV (genome copies number variation) and detects；Parameter is acquiescence.
4.6 pairs of library frequency corrections and variation annotation：For the detection mutation in single library, calculates the mutational site and exist
The average frequency of mutation in two libraries, as the frequency of mutation after the correction of the mutation.Detection variation is carried out using ANNOVAR softwares
Annotation, notes content includes base mutation, amino acid mutation, functional mutant etc..
In the present embodiment kind, why it is parallel build library, be because DNA is after being divided into two parts, be not random distribution, therefore
Background is reduced after being divided to two parts, improves detection.The result difference in two libraries is larger, also exactly illustrates this problem, without
It is method inaccuracy.Using 2 libraries, the reason is that amount of DNA is natively few, it is divided into the more parts of risks that will increase experiment loss.
Reference site
1.BWA：http://bio-bwa.sourceforge.net/
2.Picard：https://broadinstitute.github.io/picard/
3.GATK:https://www.broadinstitute.org/gatk/
4.Mutect:http://www.broadinstitute.org/cancer/cga/mutect
5.CONTRA:http://contra-cnv.sourceforge.net/
6.ANNOVAR:http://annovar.openbioinformatics.org/en/latest/
7.SAMtools:http://samtools.sourceforge.net/
5. sequencing result is analyzed
50 patient mutations' detection results are as shown in table 2 below, the results show that all sample standard deviations have drug resistance prominent in the present embodiment
The positive findings of change detect, and the result of double library detections also shows the nonunf ormity of mutation sequencing read, this is
Since the abundance of mutant DNA is extremely low, normal DNA abundance will produce very big detection background, and double library construction strategies, can be with
The detection background in single library is reduced, is conducive to the detection of mutation, all patients' detection mutation are 22 total, and mutation is summarised in
It is listed in table 3.
Table 2：50 patient mutations detect result
The mutation detection result of table 3. is summarized
5. statistical analysis
Further using the method for capture sequencing, retrospective mutation is carried out to 50 patient's biopsy samples and is verified, hair
Detection mutation is consistent with mutation in peripheral blood in existing biopsy sample.
The blood sample of 50 normal controls has carried out the mutation that related locus is not detected in Parallel testing, illustrates these mutation
It is sufficient to distinguish colorectal cancer Cetuximab drug resistance individual and normal individual.
According to the above results, inventor devises a kind of capture chip of simplification, the capture chip include for table 2 or
The probe of listed gene loci in table 3.Specific design method, which is sketched, is：Sequence based on listed gene loci in table 2 or 3
Row design the probe of a 100bp, from front to back successively according to reference sequences per~100bp progress probe coverings every 10bp
Design.Above-described embodiment is repeated, and in step 3.1, by catching after Quality Control qualification in library after amplification and using these simplification
Obtain probe, the specification provided with reference to chip manufacturer (Roche) carries out hybrid capture and carries out follow-up sequencing steps, obtained with
The identical result of above-described embodiment.It therefore, can using simplified sequence in the case where reducing synthesis, experiment, sequencing cost
It achieves the object of the present invention.
It is regardless of and is limited to any theory, the present inventor is by the optimization that is carried out to reaction system and selection to suitable gene order
The detection of combination realizes the detection of the drug resistant trace amount DNA of colorectal cancer Cetuximab (nanogram level) sample.To reaction system
Optimize including polymerase be selected as Phusion rather than KAPA is serial, in addition also have be added DMSO, increase trace
DNA degree of detection.The check experiment of parallel progress, does not reach the detection result in the embodiment of the present invention.
Claims (10)
1. a kind of method of colorectal cancer Cetuximab drug resistance trace amount DNA abrupt climatic change, the method includes the steps：
(1) DNA is extracted respectively from examined samples and check sample；
(2) DNA is built into library respectively；
(3) library is enriched with respectively；
(4) library of the enrichment is captured respectively using capture probe, obtains capture dna；
(5) capture dna is sequenced, obtains sequencing result；
(6) by the sequencing result of the examined samples and the check sample, mutational site is detected.
2. method of claim 1, the method further includes (7) for detected mutational site, calculates the mutational site
The frequency of mutation.
3. the dissociative DNA is divided into two parts in step (2) while building two libraries, in step by the method for claim 2
(7) for detected mutational site in, the mutational site average frequency of mutation in two libraries is calculated, as the mutational site
The frequency of mutation after correction.
4. method of claim 1, in step (6), for multiple examined samples, mutational site detection is as follows：
Obtain the vision response test μ of the capture probe of each examined samples and the locus specificity mistake of the multiple examined samples
Rate e；
It calculates in examined samples, sequencing read number occurs for mistake expected from mutated site, which is divided into whole mistake and surveys
Sequence read desired value NμWith the wrong desired value N of locus specificitye, formula is as follows：
Nμ=D* μ
Ne=D*e
Wherein, total sequencing depth of D mutated sites in examined samples,
It assesses in examined samples respectively, the mutation sequencing actually detected several N of read and mistake generation sequencing read number desired value are on the berth
Confidence level p in pine distributionNμAnd pNe, formula is as follows：
pNμ=1-ppois (N, Nμ)
pNe=1-ppois (N, Ne)
If pNμAnd pNeIt is less than conspicuousness threshold value, such as 0.05 simultaneously, and compares mutation sequencing read number≤2, the i.e. point mutation
Really to detect mutation.
5. the method for claim 4 calculates the vision response test μ of the capture probe：Formula is as follows,
Wherein n is the base number of capture probe, and i is some site in capture probe, MiFor the false bases number in the sites i, DiFor i
The total depth in site.
6. the method for claim 4 calculates the locus specificity error rate e：Formula is as follows,
Wherein, m is check sample number, and j is the sample for specific site occurring, base mutation (such as G sports T) occurring, MjFor in j samples
In this, the sequencing read of the base mutation type supports number, DjFor total sequencing depth of the mutated site in j samples.
7. the method for claims 1 or 2, the gene order of listed gene during capture probe is for table 1 in step (4)
The probe of listed gene mutation in probe, option list 2 or table 3.
8. implementing to be mutated inspection according to the colorectal cancer Cetuximab drug resistance trace amount DNA of the method for any one of claim 1-7
The device of survey, described device include：
DNA sample extraction module, for extracting DNA respectively from examined samples and check sample；
Library module is built, for the DNA to be built library respectively；
Library enrichment module, for the library to be enriched with respectively；
Trapping module obtains capture dna for capturing the library of the enrichment using capture probe；
Sequencer module obtains sequencing result for the capture dna to be sequenced；
It is mutated check-out module, according to the sequencing result of the examined samples and the check sample, detects mutational site.
9. a kind of capture chip, the capture chip includes the capture probe for the gene order of listed gene in table 1, is appointed
It is the probe for gene mutation listed in table 2 or table 3 to select the capture probe.
10. being used to indicate the gene order combination of cancer of pancreas, the assortment of genes includes the gene sequence of listed gene in table 1
Row, listed gene mutation site preferably in table 2 or table 3.
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