CN108753967A - A kind of gene set and its panel detection design methods for liver cancer detection - Google Patents
A kind of gene set and its panel detection design methods for liver cancer detection Download PDFInfo
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Abstract
The invention discloses the preparation methods that a kind of hepatocarcinoma gene detects panel, include the following steps：Liver cancer information and its related gene are screened, gene set is obtained；Select gene target region；Design panel probes；RNA single strand probe is synthesized, genetic test panel is obtained.The genetic test panel that the present invention designs can be used for diagnosis, parting, the prognosis of liver cancer, have gene coverage wide, portable strong, be suitable for detection platform newly developed.
Description
Technical field
The invention belongs to genetic test fields, and in particular to a kind of gene set and its panel detections for liver cancer detection
Design method further relates to application of the gene set in genetic chip.
Background technology
Liver cancer is one of current clinically most common malignant tumour, according to statistics, in the world, every year about 74
Ten thousand newly-increased liver cancer cases, while having 700,000 PLC mortality cases again, it is approximately half of among these to be happened at China.Due to the height of liver cancer
Incidence and high mortality, liver cancer are worldwide all particularly paid attention to, and the onset of liver cancer rate and the death rate in China also be in by
Year ascendant trend, liver cancer are in the overall second of various malignant tumour associated death reasons, seriously endanger human health, have become
For global, severe public health problem.The occurrence and development of liver cancer are a complicated biological processes.In addition to patient
Except itself susceptible inherent cause, various extracellular factors can lead to abnormality proliferation and the differentiation of liver stem cells, and finally lead
Cause the occurrence and development of liver cancer.Fast breeding is important one of the Biological characteristics of liver cancer with transfer, the fast breeding of liver cancer with
Transfer is to lead to one of the key factor that many liver cancer patient prognosis are poor, five year survival rate is low.Therefore, research causes liver cancer fast
The reason of speed proliferation is with transfer, the molecular mechanism for disclosing onset of liver cancer have extremely important work to improving liver cancer patient prognosis
With.Primary carcinoma of liver early stage is generally without any symptom, once there is clinical manifestation, the state of an illness has been enter into middle and advanced stage mostly, and dislikes
Property degree is high, progress is fast, poor prognosis, invasion are strong, therefore the diagnosis of liver cancer, especially early diagnosis are clinic diagnosis and prognosis
Key, and determining crucial mutator will lay the foundation for the diagnosis of patient, parting and targeted therapy.
In recent years, with the rapid development of molecular biology and the appearance of high throughput sequencing technologies, for epigenetics
Cognition and research it is also more and more.By carrying out high-flux sequence to the genome in biological sample come the technology of diagnosing liver cancer
There is tremendous development, but the detection of existing liver cancer has the following defects：1. detection content is single, function is limited.At present
Detection pertains only to part albumen to be observed or gene.For genetic test, based on gene capacity gauge and sequencing cost, covering
Gene it is also fewer, only focus on mostly targeted drug treatment related locus, other related locus are less, for scientific research explore and
The discovery feature of novel targets is limited.2. gene collection method is simple.Current majority panel designs are only for drug guide, label
And limited literature's store, based on large scale sequencing platform result, public database and Text Mining Technology comprehensive analysis compared with
It is few.3. current panel design synthetic method techniqueflow is single, always there is a region that can not be designed by fixed algorithm, therefore for
Inevitably there is omission in some important sites.4. applicable platform is few, developed panel is detected, has one on common platform
Detection architecture scheme is covered, and for new detection platform, such as BGISEQ-500, not yet there is suitable detection scheme at present.5.
Information analysis method flow is cumbersome.The analysis of biological information of lower machine data is needed to assess step by step, substep is completed as a result, step
Rapid cumbersome, the time is long, portable not high, cannot meet current analysis demand.
Invention content
In order to make up the above-mentioned deficiency of existing liver cancer related gene detection technique, inventor develops a kind of new liver cancer base
Because collecting panel detection design methods, gene panel contains all types gene detected for liver cancer, gene coverage
Extensively, it can be used for each stage of the liver cancer genesis and developments such as diagnosis, treatment, the prognosis from liver cancer risk assessment to liver cancer, and
It is portable strong, it is suitable for the sequencing instrument of domestic market or detection platform newly developed.Specifically, the skill of the present invention
Art scheme is as described below.
A kind of preparation method of hepatocarcinoma gene detection panel, includes the following steps：
1) liver cancer information and its related gene, are screened, local data base is established：The pairing of same liver cancer patient is collected respectively
Sample, the paired sample are that solid tumor mass sample/haemocyte or normal structure (cancer beside organism) sample match, and are used
NGS sequencing technologies to carrying out genetic test to paired sample, are compared by analysis respectively, select the gene of the frequency of mutation >=5%
Region；
2) it, selects to disclose liver cancer information and its related gene from open source literature resource, it is described to disclose liver cancer letter
Breath and its related gene include：High frequency mutant gene in public database using standard, treatment guidelines, drug label and is led to
With the liver cancer treatment related gene in database, it has been disclosed that reported in the literature and liver cancer molecule parting, treatment, prognosis, induction
Relevant gene；
3), above-mentioned liver cancer information and its related gene are merged, de-redundancy, and passes through NCBI office name, HGNC
Approved Official Symbol systems determine standard gene name, obtain liver cancer detection chip gene set, which can
For instructing the preparation of liver cancer detection chip；
4), gene target region of the selection for probe design：For the gene collected in step 1) and step 2), if
Specific variable position is specified, then according to specific gene loci overlay area come selection target region；Position is relatively concentrated
Or intensive gene region, then select exon as target area；For the important gene highly relevant with liver cancer, then select
The region of whole variable sheer types is as target area；
5), the gene target region both ends selected in step 4) are extended, default-length 50bp, merges all selection areas
De-redundancy behind domain, ultimately forms the bed files of probe design, which includes chromosome numbers, the target area of target area
The initial position in domain, the final position of target area, self-defined information；
6), panel probes design：According to the target area selected in step 4), probe can be designed from human genome
The design section of probe can be designed by being found in data set, and probe is generated according to design section and probe design parameters；
7) probe design evaluation：The important site and target complete region and step highly relevant with liver cancer that will be filtered out
6) the probe design section selected in is compared, and calculates coverage and target complete region that probe covers the important site
Coverage, calculation formula is：Reading long number mapping reads/ target sequencing reading length numbers target in coverage=comparison
Reads meets the covering demand of whole coverage >=90%, important site coverage >=99% as crossed, then can enter in next step
Panel is synthesized；As unqualified, then proper probes need to be selected in close-proximity target zone；
8) panel is synthesized：Fixed extension increasing sequence is added at the probe both ends designed in step 7), and synthetic DNA is single-stranded,
PCR amplification is transcribed into rna probe, adds biotin labeling, obtains hepatocarcinoma gene detection panel.
The screening process of above-mentioned liver cancer information and its related gene can record various data by perl script flow
Enter, standardizes filtration treatment, finally export alternative gene lists.
In one embodiment, above-mentioned steps 2) described in the collection of high frequency mutant gene in public database be logical
It crosses and the detection data for detecting liver cancer patient in public database is filtered, high frequency mutant gene is selected, under screening technique is
TCGA COSMIC databases are carried, the gene mutation frequency of liver cancer patient is calculated, the frequency of mutation of gene is calculated, are sorted
After filter, default parameters be preceding 20 genes；In the same way, the testing number of liver cancer sample is selected in ICGC databases
According to, including tetra- partial data of LIRI-JP, LINV-JP, LIHC-US, LICA-FR, LIHM-FR, calculates the frequency of mutation of gene,
It is filtered after sequence, default parameters is preceding 20 genes.
In one embodiment, above-mentioned steps 2) described in apply standard, treatment guidelines, drug label and conventional data
The collection of liver cancer treatment related gene in library is from database PGKB, NCCN, US.FDA drug, CFDA, PGKB application mark
Liver cancer treatment related gene is collected in standard, treatment guidelines, drug label and Universal Database.
In one embodiment, above-mentioned steps 2) described in report in open source literature with liver cancer molecule parting, treatment,
Prognosis, the collection for inducing relevant gene are the important genes filtered out by prior art text mining in document, and text is dug
The key parameter of pick includes cancer types, pathogenesis, risk of cancer, prognosis, detection method, Molecular Detection type, treatment side
Method establishes dictionary by keyword, and the "and/or" between keyword is selected to contact, by script to NCBI Pubmed documents
It is excavated, is finally filtered out in document and liver cancer molecule parting, the related, prognosis for the treatment of, induction phase from selecting for text mining
The important gene of pass.
One gene may belong to parting, treatment, prognosis, induce related multiple attributes and the same gene in difference
Source have different names.Above-mentioned steps 3) described in " de-redundancy " be to these situations carry out deduplication, finally screen
Gene in list of genes has and only will appear primary.
Above-mentioned steps 5) described in " de-redundancy " be to gene region deduplication, what is finally obtained in this way is one and is positioned at
Unduplicated region collection on chromosome.
Preferably, above-mentioned steps 6) described in can to design probe data collection be by Alu repeat regions (the i.e. Alu of genome
Repeat), the multiplicity in tandem repeat region (i.e. tandamrepeat) and pseudogene (i.e. pesudo gene) it is high or
Reject the data set that can carry out capture probe design formed later in the low region of assembling quality.
Above-mentioned steps 6) in when carrying out the design of panel probes, the design covering of the probe of design section is weighted, is covered
Degree weighted data is to predict some regions according to sequencing data of whole genome there are excessively high or too low depth, to this part
Region carries out quantization label, to achieve the purpose that the probe that final design goes out can uniformly capture target area, wherein weighted formula
It is as follows：
In formula, x1, x2, x3 ... xk is above/or less than average probe depth data, f1, f2, f3 ... fk are each
The corresponding weights of depth of probe data, k are excessively high/too low deep statistical quantity,It refer to average probe depth.
Above-mentioned depth refers to probe overburden depth, and probe covering means that depth is low less.
In a preferred embodiment, above-mentioned steps 8) in the rna probe be single stranded RNA probe.
Preferably, the hepatocarcinoma gene detection panel is fixed on the silicon chip or slide of genetic chip；Or hepatocarcinoma gene
Detecting panel uses liquid phase reactor pond, RNA single strand probe that can directly participate in subsequent reactions, need not be fixed on solid phase carrier.
Another aspect of the present invention is to provide a kind of genetic chip, liver cancer detection device or kit comprising
Hepatocarcinoma gene obtained by the above method detects panel, for example is made of hybridization probe.
Above-mentioned genetic chip, liver cancer detection device or kit can be used for detecting the genome in biological sample, with
Just diagnosis, parting, the prognosis of liver cancer are used for.
Another aspect of the present invention is to provide a kind of hepatocarcinoma gene detection panel, includes shown in table 1 for detecting
378 genes gene set.
Preferably, the gene set includes following genes：TP53, CTNNB1, AXIN1, ARID1A, APC, FAT3, NAV3,
KMT2D, TAF1, NCOR1, RB1, SETD2, TSC1, TSC2, ALK, CARD11, CHD2, NFE2L2, NOTCH4, PIK3CG,
SMARCA4, CCND1, PTEN.
Obviously, above-mentioned gene set can detect the target of panel as hepatocarcinoma gene, be used to prepare genetic chip.
Hepatocarcinoma gene detection panel prepared by the method for the present invention has the following advantages that：
1. detection performance improves.The data source being related to is extensive, other than autonomous sequencing assay result, also by mesh
Liver cancer detection data carries out calculating sifting in preceding generally acknowledged frequently-used data library, by text mining to having delivered the high pass in document
Amount detection data is screened, and the gene coverage of final choice is wide, high with liver cancer correlation.The exploitation of the technology is not limited to
In the treatment period of liver cancer, each stage of liver cancer genesis and development can be included, from liver cancer risk assessment, to liver cancer diagnosis,
Treatment, prognosis can include all types gene of liver cancer detection.
2. being more suitable for specific crowd.The present invention contains the testing result of high-flux sequence, which suffers from according to China
The genetic mutation testing result of person filters out high frequency mutant gene, therefore the gene panel designed is more suitable for compatriots'
Detection.
3. controllability is stronger, coverage higher.The method of the present invention is stronger to the probe design Modulatory character of panel,
The probe of design is set to better cover important area by the screening of different parameters, target area entirety coverage is also higher.
4. testing cost is low, the scope of application is wider.The panel that the present invention develops is suitable for the sequencing instrument of independent development,
For the detection method of current commercial company monopolization, the detection and analysis of panel detection platforms of the invention to gene
Cost is lower, great popularization.
5. analysis is convenient, portable strong, operability is strong.The software package that information analysis in the technology is formed can pacify
Loaded on multiple platforms, can be completed from lower machine data to various one steps of variation result, analysis time greatly shortens, and strives for for patient
The valuable detection and analysis time.
Description of the drawings
Fig. 1 shows the gene mutation frequency of panel detection of the present invention for 110 liver cancer patients.
Fig. 2 shows that the mutation (variation) of panel detection of the present invention for 110 liver cancer patients is distributed.
Specific implementation mode
The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for
The bright present invention is not for restriction the scope of the present invention.
Gene set (gene set) herein is all and the relevant gene sets of liver cancer or the assortment of genes.
A word after genetic test panel is high-throughput amount genetic test and gene sequencing grows up, it is
Refer to and the corresponding probe of several genes is designed on same capture chip to capture target dna and be used for subsequent in the detection
Gene sequencing.It is more than in the detection and detects a site, a gene, but detect multiple sites, multiple genes, more simultaneously
A site, these sites and gene needs are selected and are combined according to a standard, and a detection panel is needed.
Herein, term " genetic test panel ", " gene panel " or " panel " indicates that identical meaning can be with
It is used interchangeably, refers in particular to combine as the rna probe set or hybridization probe of polygenes detection means.
Gene panel refers in particular to count what target gene either genome area was detected for a small amount of in the application
A kind of probe Acquisition Scheme.For being detected compared to full-length genome, the gene panel of the application can only capture a small amount of target area
To be detected, it is related to the related gene and its mutator in liver cancer each stage.The gene panel of the application is by detecting liver
The hybridization probe of cancer information and its related gene forms, and the silicon chip or glass of genetic chip may be used in common solid phase carrier
Piece.It is of course also possible to be the probe reaction pond of liquid phase, various reactants and probe can be completed to reaction in reaction tank,
This is not specifically limited.In a kind of embodiment of the application, the gene panel for liver cancer detection is exactly to use liquid phase reactor
Pond.
Herein, term " local data base " refers to the mechanism of independent development genetic test platform by voluntarily collecting life
Object sample (includes liver cancer patient and the blood sample of healthy population from each stage, tumor tissues, cancer beside organism, just
Often tissue etc.), the gene information database established by gene sequencing.
Herein, term " liver cancer information and its related gene " includes not only gene information such as base sequence, dyeing
Body number, position, repetitive rate etc. further include and the relevant information of liver cancer such as liver cancer parting, developing stage, treatment, prognosis, sample
This type etc..
Herein, term " important gene " refers to and liver cancer parting, treatment, prognosis, induction (or driving) height phase
The gene of the frequency of mutation >=5% is detected in the gene of pass, especially paired sample.
Herein, term " important site " refers to brighter to functional studies such as liver cancer treatment, prognosis, access, frequencies
True site.
The important site coverage of 378 liver cancer information of gene panel probes pair and its related gene that the present invention is built
More than 99%, target complete area coverage is more than 90%.
In a specific embodiment, the number of probes for detecting 378 liver cancer information and its related gene is 3.9 ×
104When, the important site coverage of 378 liver cancer information of probe pair and its related gene is 100%, and target complete region is covered
Cover degree is 95%.
The liver cancer such as diagnosis, treatment, the prognosis from liver cancer risk assessment to liver cancer are covered due to the gene panel of the application
Each stage, therefore can be used for prepare respective stage genetic chip, gene assaying device or kit.
As a preferred embodiment, the preparation method of the gene panel of the application mainly includes the following steps that：
1. the screening of liver cancer information and its related gene.Liver cancer information and its dependency basis including collecting at least three aspects
Cause, in a first aspect, collecting the sequencing of liver cancer patient sample obtains in local data base liver cancer information and its related gene；Second party
The liver cancer information disclosed in public database and its related gene are collected in face；The third aspect collects the liver cancer letter disclosed in document
Breath and its related gene；The liver cancer information and its related gene collected in terms of three are merged, de-redundancy, and passed through
HGNCapproved Official Symbol determine standard gene title, form gene set；
2. detecting target area selection.Based on the gene set obtained in step 1, selection target region；Selected
Target area both ends extend 50-100bp, merge de-redundancy after whole selection regions, ultimately form the bed designed for probe
File, the bed files include chromosome information, start position information, final position information；Wherein, in selected target area
Domain both ends extend, and are to ensure that the when of designing probe can cover the region at target area most both ends, generally extending 50-
100bp, the specific size that extends is depending on the panel area sizes finally designed, a kind of embodiment of the application
In, the gene panel for liver cancer detection is to extend 50bp；
In one embodiment, the method in selection target region specifically includes, and first, for the base of known variable position
Cause, according to the overlay area selection target region of known variable position；Second, for position compared with concentration or intensive gene regions
Domain then selects its exon as target area；Third selects the region of its whole variable sheer type for important gene
As target area.
3.panel probes design.According to human genome, the probe design section that can carry out capture probe design is formed
Database；According to the location information for being formed by each target area in bed files, each target area is found in the database
Corresponding probe design section；Probe is generated according to probe design section and probe design parameters；
It is preferred that the design coverage to generated probe is weighted, design coverage weighted number is surveyed with mankind's full-length genome
Ordinal number it is predicted that the depth of each probe design section be foundation, specifically, the sequencing data of each probe design section is predicted
Depth carry out quantization label, the probe of the probe design section higher or lower than mean depth is weighted, so that finally
The probe designed can uniformly capture each target area；For example, to the probe design section less than mean depth, increase its power
Weight so that probe can also have preferable capture ability and effect to the region of low depth, and it is uniform to reach each target area with this
The effect of capture；
4. probe is assessed：For important site and target complete region, the probe design section of itself and selection is compared, is counted
Calculate the coverage in the important site of covering of each probe and the coverage to target complete region；
Calculation formula is the reading long number in coverage=comparison/target sequencing reading length number,
Important site coverage >=99%, the probe of target complete area coverage >=90% is selected to be synthesized for panel；
If probe coverage cannot meet requirements above, proper probes are reselected near probe design section；
5.panel is synthesized：Fixed extension increasing sequence is added at designed probe both ends, synthetic DNA is single-stranded, PCR amplification,
Then it is transcribed into rna probe again, and adds biotin labeling on rna probe, obtains the gene panel for liver cancer detection.
Preferably, the local data base in step 1 refers to the liver cancer and its phase by the way that the liver cancer patient sample obtained is sequenced
The database of the gene region of correlation gene, the preferably frequency of mutation >=5%.
Preferably, the public database described in step 1 includes：TCGA COSMIC databases and ICGC databases；Using
Standard, treatment guidelines, drug label and/or Universal Database.
It is preferred that collecting highest at least preceding 20 genes of gene mutation frequency in TCGA COSMIC databases, ICGC is collected
Highest at least preceding 20 genes of gene mutation frequency in database.
The application standard, treatment guidelines, drug label and Universal Database include but are not limited to PGKB, NCCN,
NCBI PUBMED、US.FDA drug、CFDA。
In one embodiment, the method for liver cancer and its related gene disclosed in document is collected in step 1 includes：Text
This excavation filters out liver cancer and its relevant gene in article, include but are not limited to liver cancer molecule parting, treatment, prognosis,
The related gene of driving；The key parameter of text mining includes but are not limited to cancer types, pathogenesis, risk of cancer, pre-
Afterwards, detection method, Molecular Detection type, therapy；Dictionary is established by key parameter, and selects the connection between keyword
System, excavates NCBIPubmed documents；Finally obtained from the document of screening include but are not limited to liver cancer molecule parting,
The related gene for the treatment of, prognosis, driving.
The gene panel that the present invention obtains is applicable in all types gene that target contains liver cancer detection, gene coverage
Extensively, it can be used for each stage of the liver cancer genesis and developments such as diagnosis, treatment, the prognosis from liver cancer risk assessment to liver cancer；Also,
The gene panel of the application can be adapted for the sequencing instrument of domestic market, and the cost of genetic test and analysis is lower.For
Comprehensive, systematization the detection and research of liver cancer is laid a good foundation.
The technique effect of the embodiment verification present invention is sequenced and analyzed as follows with Panel.The percentage arrived involved in embodiment
Content all refers to mass percentage unless otherwise indicated (for example being explicitly indicated as volume basis ratio or ratio).
Embodiment 1 Panel sequencings
1) sample collection
According to《Chinese primary carcinoma of liver diagnosis and treatment specification》Acquisition standard, 110 liver cancer patients of medical institutions pair carry out samples
This acquisition.
2) prepared by the extraction of sample DNA, library
Every 1 sample of liver cancer individuation genetic test is using hepatic carcinoma FFPE as experimental group, the blood of same period acquisition
Sample is as a control group, it is contemplated that and label connector, hybridization and the influence of data volume utilization rate are assessed building library quantity,
Confirmation once builds library sample 4 to being best.FFPE nucleic acid extraction kits and blood cell extracts kit is used to carry out respectively
Sample extraction, and carry out quantitative detection using Qubit DNA fluorescent quantitation instruments and Qubit dsDNA HS Assay kit, it is desirable that
Extracting concentration is assessed, and confirms 10ng/ μ L, yield is best more than 200ng.
The DNA sample of 200ng is respectively asked for, addition is interrupted repairs reaction mixture, reagent component with end：DNA fragmentation
Enzyme (0.4U/ μ l)+Klenow segments (5,000U/mL)+polymerase (10000U)+5:1 dATP：DNTP (1mM)+DNA fragmentation
Change enzyme buffer liquid (10 ×), after mixing well centrifugation, is reacted by following procedure：37 DEG C of 20min, 65 DEG C of 15min, 4 DEG C of guarantors
It holds.
After reaction, label connector and connection reaction mixture are added in former pipe：ATP (100mM)+polyethylene glycol
8000 (50%)+T4 polynucleotide phosphokinase buffer solutions (10 ×)+nuclease-free water+ligase (600U/ μ l), carry out with
Lower reaction：23 DEG C of 60min, 4 DEG C of holdings.
It is purified after reaction, products therefrom is dissolved in nuclease-free water, is directly carried magnetic bead and is carried out PCR amplification
Reaction, the amplified reaction use KAPA-PCR reaction solutions, and upstream and downstream PCR primer is added and sample is reacted.Reaction system
Component includes：Ammonium sulfate, magnesium chloride, dithiothreitol (DTT), trishydroxymethylaminomethane, PCR enzymes.
Reaction process is 95 DEG C of reaction 5min first；Enter back into circulation step：98 DEG C of reaction 15s, 60 DEG C of reaction 10s, 72
DEG C reaction 40s, the step cycle 7 times；Then 72 DEG C of reaction 5min；Last 4 DEG C of holdings.
PCR product is quantified using Qubit HS, it is desirable that PCR concentration is more than 25ng/ μ L, and yield is more than 550ng.
3) hepatocarcinoma gene probe specificity hybrid capture
Using 4 FFPE samples as one group, 4 blood samples different blood sample in i.e. 4 sources is as one group, this example
Blood sample be haemocyte in whole blood sample, each sample input 500ng adds up to 2000ng to carry out 2 groups of fragmentations sieves respectively
Choosing, the product after screening are dissolved in 16 μ L nuclease-free waters, it is desirable that concentration is more than 55ng/ μ L.Then FFPE groups take 700ng with
1000ng carries out hybrid experiment after blood group takes 300ng to merge.The kit that hybrid experiment uses makes science and technology for Shenzhen Hua Da intelligence
" the target area capture common reagent " of Co., Ltd.
Hybridization step is as follows：1000ng segments screening product is settled to 18 μ L, and 8 μ L hybridization reaction solutions 1 are added, fully
Mixing, which is placed in PCR instrument, runs following procedure：95 DEG C of 5min, 65 DEG C of holdings.Temperature is down to after 65 DEG C, sample rapidly with it is miscellaneous
Reaction solution 2, hybridization reaction solution 3 is handed over to be sufficiently mixed.65 DEG C of constant temperature hybridization reactions 16 hours in PCR instrument.
Wherein, the main component of hybridization reaction solution 1 is oligodeoxynucleotide, trishydroxymethylaminomethane and ethylenediamine tetraacetic
The combination of acetic acid；The main component of hybridization reaction solution 2 is sodium chloride, sodium dihydrogen phosphate, ethylenediamine tetra-acetic acid, bovine serum albumin
In vain, the combination of ficoll 400, polyvinylpyrrolidone and lauryl sodium sulfate；The main component of hybridization reaction solution 3 is liver cancer
The combination of gene probe, 4- hydroxyethyl piperazineethanesulfonic acids, potassium hydroxide, potassium iodide, dithiothreitol (DTT) and glycerine；Refer to Shenzhen China
Pansophy makes " target area capture common reagent " specification of Science and Technology Ltd..
It waits for that after reaction, product being transferred to using in the streptavidin magnetic bead cleaned in conjunction with liquid, wherein containing
It in conjunction with liquid, is washed respectively using cleaning solution 1, cleaning solution 2 after vertical mixing 30min, product is dissolved in 45 μ L nuclease frees
In water, band magnetic bead carries out PCR reactions, is purified after reaction using purifying magnetic bead, is finally dissolved in 40 μ L nuclease frees
In water, and it is transferred to new 1.5mL centrifuge tubes.It is quantified using QubitHS, it is desirable that PCR concentration is more than 4ng/ μ L, and yield is big
In 160ng.
The streptavidin magnetic bead product of this example use DynabeadsM-280streptation, in conjunction with liquid, cleaning solution 1,
Cleaning solution 2 refers to magnetic bead product description.PCR reactions are carried out as conventional PCR reactions with magnetic bead, and reaction system includes：Sulphur
Sour ammonium, magnesium chloride, dithiothreitol (DTT), trishydroxymethylaminomethane, PCR enzymes；Each component dosage refers to routine PCR reaction.Reaction
Condition is 95 DEG C of pre-degeneration 5min；It is recycled subsequently into 14：98 DEG C of denaturation 15s, 60 DEG C of annealing 10s, 72 DEG C of extension 40s；It follows
After ring, 72 DEG C of extension 5min；Last 4 DEG C of holdings.It is purified using purifying magnetic bead, purifying magnetic bead is nanometer magnetic bead and folds
The mixture of sodium nitride, the name of product for purifying magnetic bead are AgencourtAMPureXP-medium.
4) machine is sequenced on
Gained hybrid product takes 160ng to be cyclized, and 20 μ L is taken to carry out DNB preparations after cyclisation, and DNA concentration reaches requirement
Be available on the machine sequencing, if BGISEQ-500 requires DNB concentration to be more than 10ng/ μ L, can be sequenced.Wherein, prepared by cyclisation and DNB
And upper machine sequencing steps etc. are with reference to " sequencing reaction universal kit (the joint of Wuhan Hua Da Zhi Zao Science and Technology Ltd.s production
Probe anchoring polymerization PCR sequencing PCR) " gene sequencer explanation.
2 times machine data analyses of embodiment
Paired sample of the information analysis flow for liver cancer project is analyzed, with microarray dataset (this example is such as BGI-Seq500)
Lower machine Fastq files be input, by filtering, comparison, deduplication, repeat compare, abrupt climatic change and etc. obtain most end-process
Become the basic statistics information of result and data.
Paired sample of this flow for liver cancer project is analyzed.To the fastq after machine under BGI-Seq500, first pass through
The filtration treatment of SOAPnuke filters out low quality base (≤10) ratio is more than higher than 50% or N (uncertain base) ratio
10% reading length (reads), and the front and back index of correlation of filtering is counted, including beginning data volume, valid data amount, CG contents, N contain
Amount, Q20, Q30 etc.；The clean fastq obtained after filtering are compared to mankind reference gene group hg19, the step by
Paligner is realized；Original bam files are obtained after comparison, are merged with picard and duplicate removal, to remove since PCR draws
The repetitive sequence risen；Data Quality Control, including coverage, capture rate, repetitive rate, effective depth etc. finally are carried out to obtained bam
It calculates.
Bam after Quality Control reaches a standard will be detected into row variation, including somatic SNV, omatic InDel, CNV, Fusion
And the sites chemotherapy correlation Germline.Somatic SNV and Somatic InDel first carry out pairing mutation using Varscan
Rough detection, set minimum-depth as 8, the minimum frequency of mutation is 0.5%, and obtained baseline results are using self-control filtering script
It is filtered, obtains finally being mutated list；CNV detections first carry out the rough detection of copy number variation using cnvkit, obtain every
Copy number (Copynumber) value of a gene region, is set separately the threshold value of Gain and Loss, and Gain refers to that copy number increases
Add, Loss refers to copy number reduction, and wherein Gain threshold values are that 1.1, Loss threshold values are<- 1.25, obtain final CNV results；
Fusion detections are sentenced using the inspection software of independent research by collecting the reads pair (length is read in pairing) of long Insert Fragment
Fused type that disconnected reads pair are supported simultaneously estimates its breakpoint section, is just credible fusion if breakpoint section is concentrated enough
As a result；Chemotherapy detection carries out the detection of the genotype of chemotherapy related locus using GATK4, judges chemotherapy according to each loci gene type
The effect of medicine.
3 probe capture rate of embodiment is assessed and gene set obtains
The ratio that overall sequencing amount is accounted for according to target area after sequencing analysis evaluates probe capture rate.According to actual design
The difference in region, capture rate assert that probe design is qualified generally between 30%~60%.
By passing through base for the gene of the frequency of mutation >=5% to the NGS genetic tests of 110 liver cancer patient paired samples
Because the important gene that panel detections screen includes：TP53, CTNNB1, AXIN1, ARID1A, APC, FAT3, NAV3,
KMT2D, TAF1, NCOR1, RB1, SETD2, TSC1, TSC2, ALK, CARD11, CHD2, NFE2L2, NOTCH4, PIK3CG,
SMARCA4, CCND1, PTEN.
Additionally by the screening of the databases such as TCGA, ICGC and delivering document text mining and be finally obtained one and be used for
The gene set of liver cancer detection, the gene set are related to 378 genes altogether.The list of genes obtained after final de-redundancy is as shown in table 1.
Table 1 is used for the gene set of liver cancer detection
Human genome annotation file is downloaded from UCSC, the transcript of all variable sheers of 378 genes is filtered out, carries
The exon region of transcript is taken, is integrated, overlap (overlapping) region is taken, obtains the target area of liver cancer detection, the region
Size is 1.54Mb.
50bp is respectively extended to the segment both ends of target area, the regions removal overlap retain chromosome numbers, start bit
It sets, final position, obtains target area bed files, overlay area 1.59M.
Bed file areas are gone to compare with that can design probe area database, the region that can be covered then is screened out
Design synthesising probing needle, final design probe 3.9 × 104Item, important site coverage 100%, target complete area coverage
95%.
The paired sample that the present embodiment has chosen 110 liver cancer patients is detected, and patient's positive sample is tumor tissues,
Negative sample is by blood/cancer.According to above method carry out DNA extractions, interrupt, end is repaired, first time PCR, hepatocarcinoma gene
The specificity capture of panel, second of PCR amplification obtain DNA sample to be sequenced.DNA sample quality meets on BGISEQ-500
Upper machine sequencing after machine demand.
Testing result is assessed, in 110 patients, the capture rate of panel detections is between 30-60%.Inspection
The gene mutation frequency of survey is as shown in Figure 1, mutation distribution is as shown in Figure 2.Testing result is consistent with actual conditions, it was demonstrated that this reality
The gene panel for liver cancer detection for applying example preparation can accurately be detected the liver cancer patient in each stage.
Above example verifies the gene panel detection schemes of the present invention, it should be noted that the application
Cover 378 liver cancer information and its gene panel of related gene can be used for the risk assessment in liver cancer each stage, examine
Disconnected, treatment, prognosis demand.It is appreciated that according to different detections or research and design demand, the preparation of the application can also be used
Method obtains the gene panel of more polymorphic type or particular utility, or even is not limited only to the gene panel of liver cancer detection, herein not
It is specifically limited.Under the thought without prejudice to the present invention, those skilled in the art on this basis make the present invention various
Change or modification, should equally belong to the scope of the present invention.
Claims (10)
1. a kind of preparation method of hepatocarcinoma gene detection panel, includes the following steps：
1) liver cancer information and its related gene, are screened, local data base is established：The pairing sample of same liver cancer patient is collected respectively
This, the paired sample is that solid tumor mass sample/haemocyte or normal structure (cancer beside organism) sample match, using NGS
Sequencing technologies to carrying out genetic test to paired sample, are compared by analysis respectively, select the gene regions of the frequency of mutation >=5%
Domain；
2), select to disclose liver cancer information and its related gene from open source literature resource, it is described disclose liver cancer information and
Its related gene includes：High frequency mutant gene in public database, using standard, treatment guidelines, drug label and general number
According to the liver cancer treatment related gene in library, it has been disclosed that reported in the literature related to liver cancer molecule parting, treatment, prognosis, induction
Gene；
3), above-mentioned liver cancer information and its related gene are merged, de-redundancy, and passes through NCBI office name or HGNC
Approved Official Symbol systems determine standard gene name, obtain liver cancer detection chip gene set；
4), gene target region of the selection for probe design：For the gene collected in step 1) and step 2), if clear
Specific variable position, then according to specific gene loci overlay area come selection target region；Position is relatively concentrated or close
The gene region of collection, then select exon as target area；For the important gene highly relevant with liver cancer, then whole is selected
The region of variable sheer type is as target area；
5), the gene target region both ends selected in step 4) are extended, default-length 50bp, after merging whole selection regions
De-redundancy, ultimately forms the bed files of probe design, which includes the chromosome numbers of target area, target area
Initial position, the final position of target area, self-defined information；
6), panel probes design：According to the target area selected in step 4), probe data can be designed from human genome
The design section found and can design probe is concentrated, probe is generated according to design section and probe design parameters；
7) probe design evaluation：It will be in the important site and target complete region highly relevant with liver cancer that filtered out and step 6)
The probe design section of selection is compared, and calculates probe and covers the coverage in the important site and covering for target complete region
Cover degree, calculation formula are：Reading long number mapping reads/ target sequencing reading length numbers target in coverage=comparison
Reads meets the covering demand of whole coverage >=90%, important site coverage >=99% as crossed, then can enter in next step
Panel is synthesized；As unqualified, then proper probes need to be selected in close-proximity target zone；
8) panel is synthesized：Fixed extension increasing sequence is added at the probe both ends designed in step 7), and synthetic DNA is single-stranded, and PCR expands
Increase, be transcribed into rna probe, add biotin labeling, obtains hepatocarcinoma gene detection panel.
2. the method as described in claim 1, which is characterized in that the high frequency mutant gene in public database described in step 2)
Collection be by public database detect liver cancer patient detection data be filtered, select high frequency mutant gene, sieve
Choosing method is to download TCGA COSMIC databases, calculates the gene mutation frequency of liver cancer patient, calculates the mutation of gene
Frequency filters after sequence, and default parameters is preceding 20 genes；In the same way, liver cancer sample is selected in ICGC databases
Detection data calculate the prominent of gene including tetra- partial data of LIRI-JP, LINV-JP, LIHC-US, LICA-FR, LIHM-FR
Frequency filters after sequence, and default parameters is preceding 20 genes.
3. the method as described in claim 1, which is characterized in that using standard, treatment guidelines, drug label described in step 2)
And the collection of the liver cancer treatment related gene in Universal Database be from database PGKB, NCCN, US.FDAdrug, CFDA,
Liver cancer treatment related gene is collected in PGKB applications standard, treatment guidelines, drug label and Universal Database.
4. the method as described in claim 1, which is characterized in that the receipts for the gene reported in open source literature described in step 2)
Collection is the important gene filtered out by prior art text mining in document, and the key parameter of text mining includes cancer class
Type, pathogenesis, risk of cancer, prognosis, detection method, Molecular Detection type, therapy, dictionary is established by keyword,
And the "and/or" between keyword is selected to contact, NCBI Pubmed documents are excavated by script, are finally dug from text
Selecting for pick filters out and liver cancer molecule parting, the related, prognosis for the treatment of, the relevant important gene of induction in document.
5. the method as described in claim 1, which is characterized in that it is by genome that can design probe data collection described in step 6)
Alu repeat regions, tandem repeat region, the region that multiplicity is high in pseudogene or assembling quality is low reject and formed later
It can carry out the data set of capture probe design.
6. the method as described in claim 1, which is characterized in that when carrying out the design of panel probes in step 6), to design section
Probe design covering be weighted, coverage weighted data be according to sequencing data of whole genome predict some regions exist
Excessively high or too low depth carries out quantization label to this subregion, can uniformly be captured with reaching the probe that final design goes out
The purpose of target area, wherein weighted formula are as follows：
In formula, x1, x2, x3 ... xk is above/or less than average probe depth data, f1, f2, f3 ... fk are each probes
The corresponding weights of depth data, k are excessively high/too low deep statistical quantity,It refer to average probe depth.
7. the method as described in claim 1, which is characterized in that the rna probe in step 8) is single stranded RNA probe.
8. the method as described in claim 1, which is characterized in that the hepatocarcinoma gene detection panel is fixed on genetic chip
On silicon chip or slide；Or using liquid phase reactor pond, RNA single strand probe is not fixed on solid phase carrier.
9. a kind of hepatocarcinoma gene detects panel, for detect include following genes gene set：ABCA13,CSF1R,HMGCS1,
NOTCH4、SMARCA4ABCA4、CSMD3、HN1、NPM1、SMC3ABCB1、CTNNB1、HNF1A、NRAS、SMOABCB11、
CTNND2、HNRNPA2B1、NRG3、SPAG17ABCB4、CXCL14、HOXD13、NSMCE2、SPC24ABL1、CYP1B1、HRAS、
NTRK3、SPP1ACE、CYP2E1、HUWE1、NUP107、SPRTNACVR2A、DCLK1、IDH1、OPN1SW、SRCADCY2、DDC、
IDH2、OR4F17、SRCAPADH1B、DDR2、IGF1R、OR4F3、STAT3ADIPOQ、DKK1、IGF2、OTOP1、STAT4AFP、
DLC1、IGF2R、PARP1、STK11AHRR、DMD、IGSF10、PARP4、SULF1AKT1、DMXL1、IGSF3、PCLO、
SUPT6HALB、DNMT3A、IL16、PCMTD1、SYNE1ALK、DOCK2、IL6、PDE4DIP、SYNE2AMPH、DSE、IL6ST、
PDGFRA、TAF1APC、DST、IL8、PDGFRB、TAF1LAPOB、EGF、IRF2、PEG3、TAF9AR、EGFR、IRS1、
PIK3CA、TCHHL1ARID1A、EIF2AK3、ISG15、PIK3CG、TERTARID1B、ELMO1、ITPR3、PKHD1、
TGFB1ARID2、EP300、JAK1、PKHD1L1、THBS4ARID4B、EPHA4、JAK2、PLIN2、TLR3ASPM、EPHB1、
JAK3、PLK4、TMC1ASXL3、EPS15、KAT2B、PLXNA2、TMEM170AATAD2、ERBB2、KDM6A、PML、
TMEM51ATAD3B、ERBB3、KDR、PPAPDC1B、TMEM99ATM、ERBB4、KEAP1、PRKCB、TNFATP10B、ERRFI1、
KIT、PRLR、TNFRSF1AATP8B1、EYS、KLF4、PRMT6、TP53ATR、FAM123A、KLF6、PROM1、TPD52ATRX、
FAM157A、KRAS、PTCH1、TRRAPAXIN1、FAM5C、KRT19、PTEN、TSC1BAP1、FAT3、LAMA2、PTGES3、
TSC2BAZ2A、FAT4、LCN1、PTPN11、TTC36BAZ2B、FBN1、LEPR、PTPN12、TTLL2BPTF、FBN2、LIFR、
PTPRB、TTNBRAF、FCRL1、LRP1、PTPRC、UBDBRCA2、FGA、LRP1B、PTPRD、UBE3CBRD7、FGF19、LRP2、
RAC2、UBR3BRD8、FGF3、LRTM1、RAD51C、UNC5DBRD9、FGF4、MAN2C1、RAF1、UNC80BRE、FGFR1、
MAP1B、RAMP3、URGCPBRF1、FGFR2、MAP2K1、RAN、USP25BRIP1、FGFR3、MAP2K3、RARG、
USP6C16orf62、FGFR4、MAP2K4、RASSF1、VCXC18orf34、FLCN、MAP2K7、RB1、VEGFAC9orf3、
FLNA、MAPK1、RBL2、VHLCACNA1E、FLNC、MARS、RECQL4、WRNCACNA2D4、FLT1、MCL1、RET、
WWP1CARD11、FLT3、MDM1、RHBDD2、XRCC1CBX4、FLT4、MDM2、ROS1、XRCC5CCNA2、FOXA1、MECOM、
RPL22、ZFPM2CCND1、FOXA2、MEN1、RPS24、ZIC3CCND2、FRAS1、MEP1A、RPS6KA3、ZNF226CCND3、
FZD6、MET、RSPO2、ZNF717CCNE1、G6PC、MICAL3、RUNX1、ZNF737CD3D、GAB1、MLL、RYR1、CDH1、
GALNT14、MLL2、RYR2、CDK4、GJA1、MLL3、RYR3、CDKN1A、GLB1、MLL4、SACS、CDKN1B、GLI2、MPL、
SAMD9、CDKN2A、GNA11、MTDH、SAMD9L、CDKN2B、GNAQ、MTOR、SAMHD1、CEL、GNAS、MUC1、SCN7A、
CEP85L、GOLM1、MXD1、SDK2、CFL1、GOLPH3、MYC、SELPLG、CHD1、GPATCH3、MYSM1、SERPINB3、
CHD2、GPC3、NAV3、SETD2、CHD7、GPR143、NCOR1、SETDB1、CLDN14、GXYLT1、NEDD9、SIPA1、CLU、
HDAC9、NF1、SIRT3、CNTN2、HGF、NF2、SLC10A1、COL11A1、HIF1A、NFE2L2、SLC15A2、COL6A5、
HIST1H2AL、NFKB1、SLC22A1、COMP、HIST1H2BD、NLRP1、SLC25A13、CPA2、HIST1H4B、NME1、
SMAD2、CREBBP、HLA-DQA1、NOS3、SMAD3、CRP、HLA-DRA、NOTCH1、SMAD4。
10. a kind of genetic chip, liver cancer detection device or kit, which is characterized in that including the liver described in claim 9
Oncogene detects panel.
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