CN105950709A - Kit, library building method, and method and system for detecting variation of object region - Google Patents
Kit, library building method, and method and system for detecting variation of object region Download PDFInfo
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Abstract
The invention discloses a kit, which includes a probe. The probe is fixed on a solid substrate or is free in a solution, and can specifically recognize an object region. The object region comprises one of the followings: at least one of 39 genes shown in the list 1; or a CDS region of the gene in the list 1; or a region having an upstream and downstream distance of 10-200 bp of the CDS region of the gene in the list 1. The invention further provides an application of the kit, a method for building an object region sequencing library, a sequencing method, and a method and system for detecting variation of the object region. Through the kit and/or the method and system, related gene sequences of colorectal cancer can be obtained simply and conveniently with high specificity at one time. The related gene sequences can be accurately detected and analyzed, and the detection and analysis result can assist in research of colorectal cancer.
Description
Technical field
The present invention relates to biomedical sector, concrete, relate to test kit and application thereof, more specifically, the present invention relates to
A kind of test kit, the purposes of test kit, a kind of method of target area sequencing library, a kind of sequence measurement and one of building are examined
Survey the method and system of target area variation.
Background technology
Colorectal cancer is modal malignant tumor of digestive tract, and M & M accounts for the of China's malignant tumor respectively
Three and the 4th.According to WHO latest data, the sickness rate of China's colorectal cancer is 14.2/10 ten thousand people, mortality rate 6.9/10 ten thousand
People.Colorectal cancer case 220,000 example, dead 10.9 ten thousand examples are newly sent out by China in 2008.Colorectal cancer is occurred frequently pernicious swollen as a class
Tumor, and cancer is a progressive process slowly, if it is accomplished early discovery, clinical effectively treatment, arrives
Prognosis recurrence monitoring control comprehensively, its incidence rate and mortality rate can be effectively reduced, by have huge economic benefit and
Social benefit [Jemal A, Siegel R, Ward E, et al.Cancer statistics, 2009.CA Cancer J
Clin.2009；59:225-229.].
At present the method for colorectal cancer research in early days has a lot, China's common approach be leading FOBT (be divided into five classes:
Radiometric analysis, Physical, chemical method, immuno-chemical method and haemachrome-porphyrin test), then positive is carried out colonoscopy
Look into.But major part method is still insufficient for the requirement detected in early days in terms of specificity and accuracy.
Circulating DNA is present in the extracellular dissociative DNA in the body fluid such as blood, synovial fluid, and research finds that many tumors are suffered from
Person's Circulating DNA has the biggest difference compared with normal person, due to apoptosis of tumor cells, containing certain in cancer patient's Circulating DNA
Tumor markers.The gene test diagnosis circulating dissociative DNA in recent years in tumor patient blood has become study hotspot, and research is aobvious
Show that in blood, Circulating tumor DNA is likely to become a kind of new early diagnosis of tumor and the mark of Index for diagnosis.Detection blood
Tumor-marker analyte detection in middle circulation dissociative DNA have be different from tradition tissue tumor markers detection mode, have noinvasive,
Monitoring at any time and the advantage such as early screening, and the sampling detection of circulation dissociative DNA is avoided current molecular diagnostic need to adopt
Collection cancerous tissue, as the difficulty of Specimen origin, is a kind of very promising tumor markers.
Nowadays high throughput sequencing technologies is used widely in medical research, but due to Colorectal Cancer in early days
Plasma DNA content relatively low, and there is certain error rate etc. in sequencing technologies itself, the most traditional sequence measurement without
Method differentiates order-checking mistake and tumor specimen low and medium frequency suddenlys change, and therefore exploitation easily operation, low damage, the technology of high precision are that knot is straight
The intestinal cancer difficult point that detection research field is captured in early days.
Summary of the invention
According to an aspect of of the present present invention, the present invention provides a kind of test kit, and it comprises probe, and described probe is fixed on solid phase
In substrate or be free in solution, described probe can specific recognition target area, wherein, under described target area includes
One of row:
At least one in shown 39 genes of table 1；Or the CDS region of at least one gene described in table 1；Or in table 1
The region of the upstream and downstream at least 10-200bp in the CDS region of at least one gene described.
Another aspect of the present invention provides a kind of method building target area sequencing library, and described method includes:
(1) obtaining the nucleic acid in sample to be tested, described nucleic acid is made up of multiple DNA fragmentations, and described DNA fragmentation is from fracture
Genomic DNA and/or free DNA fragmentation, described short sequence DNA fragment has flat end；
(2) add base " A " to hold to the 3 ' of described DNA fragmentation, it is thus achieved that there is the DNA fragmentation of sticky end A；
(3) jointing is in the two ends of described sticky end fragment, it is thus achieved that joint junction fragment；
(4) utilize the first primer that described joint junction fragment is carried out the first amplification, it is thus achieved that the first amplified production；
(5) utilize mentioned reagent box that described first amplified production is captured, it is thus achieved that described target area；And,
(6) utilizing the second primer that described target area is carried out the second amplification, it is thus achieved that the second amplified production, described second expands
Volume increase thing constitutes described target area sequencing library.
Another aspect of the present invention provides a kind of sequence measurement, and described method includes: check order according to above-mentioned structure target area
The method in library builds target area sequencing library；
Checking order described target area sequencing library, it is thus achieved that sequencing data, described sequencing data is by multiple reading section groups
Become；Wherein, NextSeq CN500 carries out described order-checking.
Another aspect of the present invention provides a kind of method detecting target area variation, and described method includes: (1) utilizes
Above-mentioned sequence measurement, it is thus achieved that sequencing data, filters described sequencing data, and described filtration includes getting rid of uncertain base
The ratio reading section more than 10% and/or base mass value are not more than the ratio of the base number of the 5 reading section not less than 50%；
(2) described sequencing data and reference sequences are carried out the first comparison, it is thus achieved that the first comparison result, get rid of the first ratio
The reading section identical to two reading sections of a reading section centering in result is right；By described first comparison result and described reference sequences
A part carry out the second comparison, it is thus achieved that the second comparison result；Refiltering described comparison result, described filtration includes
Remove base mismatch number in comparison and read section more than 3, it is thus achieved that in SNP, InDel, SV and CNV variation in described target area
At least one；Wherein said reference sequences is HG19, and a part for described reference sequences includes in the reference sequences of target area
The mismatched regions that each known InDel site causes.
Another aspect of the present invention provides a kind of system detecting target area variation, including,
Nucleic acid acquisition device, for obtaining the nucleic acid in sample to be tested, described nucleic acid is made up of multiple DNA fragmentations, described
DNA fragmentation is from the genomic DNA ruptured and/or free DNA fragmentation, and described DNA fragmentation has flat end；Add base A dress
Put, be used for adding base " A " and hold to the 3 ' of described DNA fragmentation, it is thus achieved that there is the DNA fragmentation of sticky end A；Joint connection,
For jointing in the two ends of described sticky end fragment, it is thus achieved that joint junction fragment；First amplification device, is used for utilizing
One primer carries out the first amplification to described joint junction fragment, it is thus achieved that the first amplified production；Acquisition equipment, for aforementioned containing spy
Described first amplified production is captured by arbitrary test kit of pin, it is thus achieved that described target area；And, the second amplification device,
For utilizing the second primer that described target area is carried out the second amplification, it is thus achieved that the second amplified production；Sequencing device, for by institute
Stating amplified production to check order, it is thus achieved that described target area variant sites information, variation includes that SNP, InDel, SV and CNV make a variation
In at least one.
The method of the present invention, is a kind of high sensitivity, high specific, high-throughout method, it is possible to auxiliary is for Colon and rectum
The scientific research of the related gene of cancer.By using a new generation's high throughput sequencing technologies, in conjunction with the test kit of one aspect of the present invention
The probe in the energy specificity capture specific gene region comprised, it is possible to the most simultaneously carry out many cases pattern detection,
And can carry out the data mining of greater depths based on same quantity of data, testing result specificity is high, has relatively low false positive
Rate, false negative rate, it can be ensured that the testing result obtained can react the real-time peripheral blood situation of person under inspection accurately.And this
Probe collection in chip is possible not only to select detection gene flexibly, moreover it is possible to along with causing the discovery of colorectal cancer new gene, add
Enter new gene, there is the highest cost performance and specific aim.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage are from combining the accompanying drawings below description to embodiment and will become
Substantially with easy to understand, wherein:
Fig. 1 shows according to one embodiment of present invention, build the flow chart of the method for target area sequencing library.
Detailed description of the invention
The present inventor, through extensively in-depth study, establishes a kind of method measuring target area variation first.Tool
For body, the present inventor, according to the information of present illness gene, devises the nucleic acid core being fixed with multiple disease specific probe
Sheet；The end of free in sample to be tested, fragmentation, to be derived from genomic DNA double chain acid molecule is added joint, goes forward side by side
Row enrichment；With nucleic acid chip, the DNA fragmentation containing joint is captured, the fragment of capture is surveyed at high-flux sequence platform
Sequence, based on known gene loci information, is analyzed sequencing result, obtains the information of target area variance.
" variation ", " variance " in the present invention, " genovariation " are generally applicable, " SNP " (SNV) in the present invention,
" CNV ", " insertion and deletion " (indel) and " structure variation " (SV) is with generally definition, but size to various variations in the present invention
Being not particularly limited, so have between these several variations has intersection, such as contaminating for large fragment even whole piece when insertion/deletion
During colour solid, fall within generation copy number variation (CNV) or chromosomal aneuploidy, fall within SV.These class form variations big
Little intersection does not hinder those skilled in the art to pass through foregoing description and performs realize the method for the present invention and/or device and reach to be retouched
The result stated.
" reference sequences " in the present invention is at least some of of known group sequence or known group sequence, this
" first ", " second " used in invention etc. only refer to for convenience of describing, it is impossible to be interpreted as instruction or hint relative importance,
Sequencing relation can not be interpreted as.In description of the invention, except as otherwise noted, " multiple " are meant that two or two
More than individual.
CDS region i.e. coding region, coding region refers to transcribe the part of messenger RNA, and it can synthesize corresponding egg
White matter.
The test kit obtaining one aspect of the present invention, the method realizing one aspect of the present invention, generally comprise target area capture
Storehouse and hybridization upper machine order-checking, the analysis of biological information of lower machine data and variation data solution are built in the design of probe/chip, trace sample
Read.
A kind of test kit, it comprises probe, and described probe is fixed on solid-phase matrix or described probe is free on solution
In, described probe can specific recognition target area, wherein, described target area includes:
At least one in shown 39 genes of table 1；Or the CDS region of at least one gene described in table 1；Or in table 1
The region of the upstream and downstream at least 10-200bp in the CDS region of at least one gene described.
Table 1
| AKAP9 | GRIN2A | PMS2 |
| AKT1 | KIT | PTEN |
| APC | KMT2C | RAF |
| ARID1A | KMT2D | RET |
| ATK11 | KRAS | RNF213 |
| ATM | MLH1 | RNF43 |
| AXIN2 | MLH3 | SMAD2 |
| BMPR1A | MSH2 | SMAD4 |
| BRAF | MSH6 | SPEN |
| CREBBP | MUTYH | TCF7L2 |
| EGFR | NRAS | TP53 |
| EPCAM | PIK3CA | TRRAP |
| FBXW7 | PMS1 | UBR5 |
In one embodiment of the invention, at least 10,20 or 30 during target area includes shown 39 genes of table 1
Gene.In one embodiment of the invention, target area includes the full gene region of shown 39 genes of table 1.The present invention
Test kit probe can the target area of specific recognition, be inventor through repeatedly collecting, repeatedly screening and test of many times
Combination obtains, and these target areas are relevant to the generation of colorectal cancer development.
Further, a length of 20-120mer of described probe.It is preferred that 50-100mer, more preferably, 60-80mer.
In one embodiment of the invention, the preparation of described probe includes the steps of determining that described target area
Reference sequences；From the beginning of one end of described reference sequences, described reference sequences obtains DNA fragmentation successively until described reference
The other end of sequence；By described DNA fragmentation and described reference sequences comparison, it is thus achieved that each DNA fragmentation is on reference sequences
Comparison number of times, filters out the comparison number of times DNA fragmentation more than 1；Get rid of G/C content not at the DNA fragmentation of 30-80%.
Wherein, a DNA fragmentation is a probe, and whole described DNA fragmentations constitute probe collection, between described DNA fragmentation
Completely overlapped, partly overlap or the most overlapping, described probe collection can cover described target area at least one times.
The reference sequences of described target area can obtain from reference to genome, such as from people with reference to genome HG19
The upper gene obtaining corresponding target area, the reference sequence of the corresponding target area described in gene composition on all of HG19
Row, HG19 can download from ncbi database.
Further, the preparation of probe also comprises determining that position on reference to genome, the described target area, obtains institute
State the reference sequences of target area, start to copy described reference sequences from first nucleotide of described reference sequences one end and obtain
Article 1, DNA fragmentation, starts to copy described reference sequences from second nucleotide of described reference sequences one end and obtains Article 2
DNA fragmentation, starts to copy described reference sequences from the 3rd nucleotide of described reference sequences one end and obtains Article 3 DNA sheet
Section, obtains follow-up DNA fragmentation the most successively until one end of N article of DNA fragmentation exceeds the other end of described reference sequences, its
In, a DNA fragmentation is a probe, and whole described DNA fragmentations constitute described probe collection, and N is that described probe concentration comprises
The sum of probe.
According to another aspect of the present invention, the present invention provides a kind of any of the above-described test kit obtaining colorectal cancer dependency basis
Because of the purposes in sequence.The test kit utilizing one aspect of the present invention can disposable, the simple and convenient and acquisition knot of high specific
The related gene sequence of rectal cancer.
According to another aspect of the present invention, the present invention provides a kind of method building target area sequencing library, described side
Method includes: (1) obtains the nucleic acid in sample to be tested, and described nucleic acid is made up of multiple DNA fragmentations, and described DNA fragmentation is from fracture
Genomic DNA and/or free DNA fragmentation, described DNA fragmentation has flat end；(2) base " A " is added to described DNA fragmentation
3 ' end, it is thus achieved that there is the DNA fragmentation of sticky end A；(3) jointing is in the two ends of described sticky end fragment, it is thus achieved that connect
Head junction fragment；(4) utilize the first primer that described joint junction fragment is carried out the first amplification, it is thus achieved that the first amplified production；(5)
Utilize mentioned reagent box that described first amplified production is captured, it is thus achieved that described target area；And, (6) utilize second to draw
Thing carries out the second amplification to described target area, it is thus achieved that the second amplified production, and described second amplified production constitutes described target area
Territory sequencing library.
This sequencing library construction method on the one hand of the present invention, is particularly well-suited to the sample sequencing library containing trace dna
Structure, in one embodiment of the invention, sample is the plasma sample containing micro free DNA fragmentation, comprises extremely trace
Target dissociative DNA fragment, the first amplification makes the amount of nucleic acid can meet the demand of chip/probe hybrid capture, and because of chip
Hybrid capture can be lost a certain amount of nucleic acid, and the second amplification can make the target fragment acquisition under capture again expand to meet upper machine
Order-checking and the requirement of Quality Control detection.This library constructing method of the present invention be particularly well-suited to always to dissociate nucleic acid be not less than 10ng or
Person's conventional organization genomic DNA is not less than the sequencing library of the sample of 1 μ g and builds, and utilizes this method structure on the one hand of the present invention
The library, target area built, the lower machine quality of data after order-checking is high, is beneficial to follow-up accurate inspection based on high-quality lower machine data
Cls analysis.
In one embodiment of the invention, DNA fragmentation described in step (1) is had flat end and is repaired by end
Prepared by method.According to one embodiment of present invention, before DNA fragmentation is carried out end reparation, may further include purification
The step of DNA fragmentation, thus so that follow-up end reparation is prone to carry out.According to embodiments of the invention, DNA fragmentation is entered
Row end reparation can utilize Klenow fragment, T4DNA polymerase and T4 polynucleotide kinase to carry out, wherein, and described Klenow
Fragment has 5 ' 3 ' polymerase activities and 3 ' 5 ' polymerase activities, but lacks 5 ' 3 ' 5 prime excision enzyme activities.Thereby, it is possible to side
The most exactly DNA fragmentation is carried out end reparation.According to embodiments of the invention, it is also possible to farther include to repair through end
The step that multiple DNA fragmentation is purified, it is possible to carry out easily subsequent treatment.
Further, the 3 ' ends at the DNA fragmentation repaired through end add base A, in order to obtain and have viscosity end
The DNA fragmentation of end A.According to one embodiment of present invention, it is possible to use Klenow (3 ' 5 ' exo-), i.e. have 3 ' 5 ' outward
Cutting the Klenow of enzymatic activity, the 3 ' ends at the DNA fragmentation repaired through end add base A.Thereby, it is possible to easily and accurately
Base A is added to 3 ' ends of the DNA fragmentation repaired through end.According to embodiments of the invention, it is also possible to wrap further
Include the step that the DNA fragmentation with sticky end A is purified, it is possible to carry out easily subsequent treatment.
Further, it is possible to use thermal starting taq archaeal dna polymerase carries out PCR amplification to the purpose fragment through conversion.
According to embodiments of the invention, the kind of thermal starting taq archaeal dna polymerase is not particularly limited, specifically showing according to the present invention
Example, thermal starting taqDNA polymerase can be r-taq polymerase, and thus PCR amplification efficiency is high, the used time is few.
In one embodiment of the invention, described first primer sequence such as SEQ ID NO:1 and SEQ ID NO:2 institute
Show；Described second primer sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4.
Wherein in SEQ ID NO:2, " NNNNNNNN " represents sequence label, and described sequence label has SEQ ID NO:5-8
Sequence shown at least one of.
In one embodiment of the invention, described method has the feature that described samples sources is in human or animal；Institute
Stating target area is colorectal cancer related gene region.
According to an aspect of of the present present invention, the present invention provides a kind of sequence measurement, and described method includes: according to the present invention one side
The sequencing library construction method in face builds target area sequencing library；Described target area sequencing library is checked order, it is thus achieved that
Sequencing data, described sequencing data is made up of multiple reading sections；Wherein, NextSeq CN500 carries out described order-checking.
Further, sequencing technologies can use second filial generation sequencing technologies or third generation sequencing technologies to carry out.Those skilled in the art
It should be understood that described order-checking platform can also use the Hiseq2000/2500 platform of Illumina, Life
The Ion Torrent platform of Technologies, single-molecule sequencing platform etc..In one embodiment of the invention, shellfish is used
Auspicious and Kanggong department NextSEQ CN 500 checks order platform.
According to an aspect of of the present present invention, the present invention provides a kind of method detecting target area variation, and described method includes:
(1) arbitrary sequence measurement of the aforementioned present invention is utilized, it is thus achieved that the target area sequencing data of target sample, to described sequencing data
Filtering, described filtration includes getting rid of the uncertain base ratio reading section more than 10% and/or base mass value is not more than 5
Base number ratio not less than 50% reading section；(2) described sequencing data and reference sequences are carried out the first comparison, it is thus achieved that the
One comparison result, the reading sections getting rid of two reading sections of in the first comparison result one reading section centering identical are right, wherein said
Reference sequences is HG19；A part for described first comparison result Yu described reference sequences is carried out the second comparison, it is thus achieved that second
Comparison result；Refiltering described comparison result, described filtration includes getting rid of base mismatch number in comparison and is more than 3 readings
At least one section, it is thus achieved that in SNP, InDel, SV and CNV variation in described target area；Wherein, described reference sequences
A part includes the mismatched regions that each known InDel site in the reference sequences of target area causes.Here, described second
Comparison is Local Alignment, and the first comparison is conventional overall comparison, may utilize but is not limited to the softwares such as SOAP or BWA and gives tacit consent to according to it
Setting is carried out, it is thus achieved that the first comparison result, and the first comparison result includes reading section matched position on reference sequences and coupling feelings
Condition information, in one embodiment of the invention, carries out the second comparison i.e. based on the first comparison result, to the gene captured
All sequences information (reads) near all known INDEL in the reference sequences that region is corresponding carries out local comparison again,
Can eliminate the mistake in the first comparison, improve the accuracy of follow-up variation detection, the second comparison may utilize GATK anharmonic ratio to soft
Part (https: //www.broadinstitute.org/gatk/) is carried out.In one embodiment of the invention, GATK is passed through
UnifiedGenotyper software detects described SNP and INDEL variation simultaneously.Utilize this variation inspection on the one hand of the present invention
Survey method, it is possible to accurately detect the low frequency sudden change that mutation frequency is 1%.
In one embodiment of the invention, described method also includes, when in detected variant sites at least it
One meet below, then judges institute test sample this as positive sample: the order-checking degree of depth not less than 10X, at least support of 3 reading sections,
Reading section in negative control sample supports number less than 2, and the mutation rate in positive control is more than 1%, and variant sites
The reading section of reading section support amount and normal control sample (negative control sample) same loci support measurer there were significant differences.Described
Positive sample refer to colorectal cancer sample.Above-mentioned decision condition is that inventor combines presently relevant database information and lot of documents
The a large amount of positive sample of report information, detection statistics and a large amount of negative sample are decided, and have statistical significance.
Specifically, described variant sites reading section support amount in sample to be tested is (negative right with normal control sample
In the same old way originally) there were significant differences for the reading section support measurer of same loci.Reading section support amount therein, can be the reading supporting this variation
The number of section, it is also possible to be the reading section supporting this variation ratio in comparison in this site reading section.
In one embodiment of the invention, using the latter to compare, described have significant difference to refer to have essence poor
Different, such as, for variant sites A in sample to be tested, reads in positive sample supports that ratio is 26/500 (to make a variation 26
Reads, total 500 reads), i.e. variation frequency 5.2% in positive sample, and the reads in negative sample supports ratio
Be 1/200 (make a variation 1 reads, total 200 reads), i.e. variation frequency in negative sample is 0.5%, then reach described
Significant difference or described substantial differences, herein refer to the significant difference having statistically, such as, can utilize
Fisher checks, and difference has significance (p≤0.05), i.e. thinks that reaching described has significant difference.
In one embodiment of the invention, another is also used to determine the algorithm of significant difference, such as treating
Variant sites A in test sample basis, the reads in positive sample supports that ratio is 7/500, and propping up in a large amount of negative sample
The ratio of holding meets specific distribution, and respectively less than 1/200, the reading section comparing variant sites A in sample to be tested supports ratio (variation frequency
Rate) with the difference of this Mutation frequency in a large amount of negative sample data sets, it is possible to use z inspection or t inspection, difference has
Significance (p≤0.05), i.e. reaches described significant difference.
Another aspect of the present invention provides a kind of system detecting target area variation, including:
Nucleic acid acquisition device, for obtaining the nucleic acid in sample to be tested, described nucleic acid is made up of multiple initial DNA fragmentations,
Described initial DNA fragmentation is from the genomic DNA ruptured and/or free DNA fragmentation；Add base A device, be used for adding base
" A " holds to the 3 ' of described short sequence DNA fragment, it is thus achieved that have the DNA fragmentation of sticky end A；Joint connection, is used for connecting
Joint is in the two ends of described sticky end fragment, it is thus achieved that joint junction fragment；First amplification device, is used for utilizing the first primer pair
Described joint junction fragment carries out the first amplification, it is thus achieved that the first amplified production；Acquisition equipment, for aforementioned arbitrary probe of comprising
Described first amplified production is captured by test kit, it is thus achieved that described target area；And, the second amplification device, it is used for utilizing
Second primer carries out the second amplification to described target area, it is thus achieved that the second amplified production；Sequencing device, for producing described amplification
Thing checks order, it is thus achieved that described target area variant sites information, variation include SNP, InDel, SV and CNV variation at least
A kind of.
In one embodiment of the invention, described first primer sequence such as SEQ ID NO:1 and SEQ ID NO:2 institute
Show；Described second primer sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4.
Wherein in SEQ ID NO:2, " NNNNNNNN " represents sequence label, and described sequence label has SEQ ID NO:5-8
Sequence shown at least one of,
In one embodiment of the invention, the system of described detection target area variation also includes:
First defecator, carries out the first filtration for the sequencing data obtaining sequencing device, it is thus achieved that first filters knot
Really, described filtration include getting rid of the uncertain base ratio reading section more than 10% and/or base mass value be not more than 5 base
The ratio of the number reading section not less than 50%；
First comparison device, for carrying out the first comparison by described first filter result and reference sequences, it is thus achieved that the first ratio
To result, the reading sections getting rid of two reading sections of a reading section centering in the first comparison result identical are right, wherein said reference
Sequence is HG19；
Second comparison device, for carrying out the second ratio by a part for described first comparison result with described reference sequences
Right, it is thus achieved that the second comparison result；
Second defecator, for described second comparison result is carried out the second filtration, described filtration includes getting rid of ratio
Centering base mismatch number more than 3 read sections, it is thus achieved that in described target area SNP, InDel, SV and CNV variation at least it
One；Wherein, the mistake that each known InDel site during a part for described reference sequences includes target area reference sequences causes
Join region.
Previously for the method detecting target area variation in one aspect of the present invention or arbitrary detailed description of the invention
Technical characteristic and the description of advantage, be equally applicable to this system on the one hand of the present invention, do not repeat them here.
Example below, is only used for explaining the present invention, and is not considered as limiting the invention.Except as otherwise explaining, below
The reagent explained the most especially, sequence (joint, label and primer), software and the instrument related in embodiment, is all conventional commercial
Product or increase income, such as builds storehouse related kit enter purchased from the NextSEQ CN 500 of Bei Rui and the Kanggong department platform that checks order
Row library construction etc..
Embodiment one design chips
1, the driving gene that in statistics OMIM data and pertinent literature, the colorectal cancer that causes of relevant single-gene is relevant is every
Individual exon 1 variation sample number, variation sample, hottest point the variation sample number at place, PI value are (to assess patient's reply frequency
Level on each exon, the every exon of PI=carries the accumulative number of patients/exon length of sudden change), and according to
PI value descending.Then using the sample of first exon 1 variation as sample database, add up other all intervals and
The number of sample database difference sample, is classified as sample intervals most for different number of samples as second and screens chip region
Between, now using two interval variation samples screening as sample database, the 3rd interval of screening in the same way,
Until sample database includes all of sample, to add up exon 1 collection, and for not screening the base in any interval
Because of all intervals, the most all it is added on chip interval.
2, based on data bases such as TCGA, ICGC, drive gene interval to remove and include the heat more than or equal to 5 samples
The interval (SNV >=5) of some variation is that candidate is interval, repeats the calculating of previous step.
3, based on data bases such as TCGA, ICGC, respectively with PI in remove the most screened interval >=30, SNV >=3
With PI >=20, SNV >=3 it is that candidate is interval, screening makes single sample database sample number reduce most intervals as first
Individual chip is interval, repeats above procedure and calculates.
4, the intervals such as fusion gene are added.
5, target area capture chip ColorectalPano is designed.ColorectalPano chip includes Colon and rectum
Driving gene (Driver Gene), high frequency mutant gene, inherited genetic and the target medicine related gene etc. that cancer is relevant, altogether 39
Individual gene, 102Kb.List of genes refers to table 1.
Embodiment two builds target area sequencing library, and idiographic flow is shown in Fig. 1.
(1) prepared by sample
1. extraction person under inspection peripheral blood 5-10mL, is stored in EDTA anticoagulant tube, and in 4-6 hour, human peripheral blood is carried out point
From；
2. plasma DNA extracts and (extracts reagent explanation with reference to QIAamp Circulating Nucleic Acid Kit
Book, carries out plasma DNA extraction)；Obtain plasma DNA (cfDNA), cfDNA may comprise from tumor cell
DNA fragmentation (ctDNA).(2) library construction
1. end reparation
Agencourt AMPure XPreagent 120 μ L, after magnetic beads for purifying, last back dissolving 42 μ L is added after reaction
DdH2O, band magnetic bead carries out next step reaction；
2. end adds A
Adding PEG/NaCl SPRI solution 90 μ L after reaction, be sufficiently mixed and carry out magnetic beads for purifying, (35-connects last back dissolving
Head) μ L ddH2O, band magnetic bead carries out next step reaction；
3. joint connects
After reaction, add PEG/NaCl SPRI solution 50 μ L, carry out magnetic beads for purifying for the first time, use 50 μ L Tris-HCl
(1mM, pH8.0) back dissolving；
Add PEG/NaCl SPRI solution 50 μ L, carry out second time magnetic beads for purifying, use 25 μ LTris-HCl (1mM,
PH8.0) back dissolving；
4. first round PCR amplification
Agencourt AM Pure XP reagent 90 μ L, after magnetic beads for purifying, back dissolving 31 μ L is added after reaction
DdH2O, Quality Control carry out chip hybridization after taking supernatant.
(3) target area capture chip hybridization
1. use the capture chip ColorectalPano-102Kb of embodiment one design, the side used according to conventional die
Method carries out hybrid capture and eluting.Finally use 21 μ L ddH2O back dissolving hybridization elution magnetic beads.
2. second take turns PCR amplification
Add Agencourt AMPure XP reagent 108 μ L, after magnetic beads for purifying, back dissolving 31 μ L EB after reaction, take
Quality Control upper machine order-checking after supernatant.
Machine order-checking in embodiment three
Sequencing library embodiment two obtained, uses Nextseq CN 500PE75 program to carry out upper machine order-checking, order-checking
Operating instruction that experimental implementation provides according to manufacturer (sees that Hangzhou shellfish is auspicious and official of health gene diagnosis company announces cBot)
Carry out upper machine sequencing procedures.
Embodiment four sequencing data analysis
1. utilize the sequencing data that the method for embodiment three obtains.
2. descend machine data filtering Reads_filter: screening to meet and analyze the reads required.Need meet two conditions:
1) number < 10% of N in reads；2) <base of 5 is less than 50% to mass value.
3. sequence alignment: Bwa aln-> sampe | samtools view | samtools sort: with the mankind with reference to gene
Group sequence alignment, obtains every reads position on chromosome and quality information.File after comparison exists with bam form；
4. deduplication MarkDuplicates.jar: comparison is attached most importance to the reads labelling with reference to the identical starting point of genome
Multiple, subsequent analysis is only used as a reads and analyzes；
5. anharmonic ratio pair: GenomeAnalysisTK.jar-T Realigner, TargetCreator,
IndelRealigner: utilize other comparison instruments to carry out comparison again pointedly ropy for early stage comparison reads, carry
High data user rate；
6. mass value correction GenomeAnalysisTK.jar-T BaseRecalibrator, PrintReads: according to
Mass value is corrected by reads feature, improves the credibility supported；
7. filter Filt_bam: remove the base mismatch number reads more than 3 in comparison, improve accuracy；
8. Quality Control QC: statistics the capture rate of chip, effective reads number, mean depth, repetitive rate, coverage and not by
The information such as the interval covered, are estimated chip design, sample process and upper machine sequencing procedure and feed back, it is ensured that quality control
Process.
9. identify SNV/InDel/SV/CNV and screen high frequency closedown site therein:
SNP variation is identified by MuTect, varScan, somVar flow process；
InDel variation is identified by gatk, varScan, somVar flow process；
CNV is identified by contra.py flow process；
SV is identified by MDect flow process；
Different inspection software and parameter is selected for different variation types
The screening parameter used is: the order-checking degree of depth >=10X, and aberration rate≤2% in negative (normally) sample, at sun
Aberration rate >=1% in property sample, supports reads number >=3 of this variation, with normal control (example in these sample to be tested data
Such as normal somatic cell) reading section support ratio there is significant difference (p≤0.05)；
10. annotation
To detection variation annotate, content includes: function, reads support number, variation frequency, amino acid variation and
Variation etc. in Cosmic, the information obtained can data base can adjust accordingly according to disease；Comment token: according to variation feelings
Condition judges that the source of disease, variation data are understood.
Five liang of example experiment sample testing results of embodiment
According to the method for embodiment 1-4, two example samples are detected.
1, testing result
Sequencing data statistical result see table 2:
Table 2
Target area coverage see table 3:
Table 3
| Sample names | Mean depth | Mean coverage | ≥4 | ≥20 |
| 16JK000003-N | 176 | 99.9% | 99.7% | 99.7% |
| 16JK000003-T | 588 | 100% | 100% | 100% |
Testing result see table 4:
Table 4
Other variation information of this gene test detection see table 5:
Table 5
Annotation: rsID: short series jump numbering in data base；FR.1:dbSNP data base includes about this
The frequency information of SNP；Fr.2: thousand people the most all check order in sample about this SNP frequency information in asian ancestry's ethnic group；
The frequency information about this SNP included in Fr.3:ESP6500 data base；Fr.4: about the frequency of this SNP in local data base
Rate information；Condel:Condel data base predicts the outcome.
2, testing result explanation
This detects in the targeting medication gene KRAS relevant to colorectal cancer, detects the prominent of a G12C
Become.This sudden change is positioned at 2 exons, and clinical application guide is pointed out, the patient of the sudden change in this site is basic to EGFR antibody drug
Invalid, and wild type patient is effective.
Embodiment described above is only to be described the preferred embodiment of the present invention, the not model to the present invention
Enclosing and be defined, on the premise of designing spirit without departing from the present invention, this area ordinary skill technical staff is to the technology of the present invention
Various deformation that scheme is made and improvement, all should fall in the protection domain that claims of the present invention determines.
Sequence table
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<120>test kit, banking process and the method and system of detection target area variation
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Claims (10)
1. a test kit, it comprises probe, and described probe is fixed on solid-phase matrix or described probe is free in solution,
Described probe specificity identification object region, wherein, described target area includes:
At least one in shown 39 genes of table 1；Or the CDS region of at least one gene described in table 1；Or described in table 1
The region of the upstream and downstream at least 10-200bp in the CDS region of at least one gene.
2. test kit as claimed in claim 1, it is characterised in that described probe is full synthetic or body outer clone synthesis,
The a length of 20-120mer of described probe.
3. the test kit of claim 1, it is characterised in that the preparation of described probe comprises the steps:
Determine the reference sequences of described target area；
From the beginning of one end of described reference sequences, described reference sequences obtains DNA fragmentation successively until described reference sequences
The other end；
By described DNA fragmentation and described reference sequences comparison, it is thus achieved that each DNA fragmentation comparison number of times on reference sequences,
Filter out the comparison number of times DNA fragmentation more than 1；
Get rid of G/C content not at the DNA fragmentation of 30-80%.
4. the purposes in obtaining colorectal cancer related gene sequence of the test kit described in any one of claim 1-3.
5. the method building target area sequencing library, it is characterised in that including:
(1) obtaining the nucleic acid in sample to be tested, described nucleic acid is made up of multiple DNA fragmentations, and described DNA fragmentation is from the base of fracture
Because of group DNA and/or free DNA fragmentation, described DNA fragmentation has flat end；
(2) add base " A " to hold to the 3 ' of described DNA fragmentation, it is thus achieved that there is the DNA fragmentation of sticky end A；
(3) jointing is in the two ends of described sticky end fragment, it is thus achieved that joint junction fragment；
(4) utilize the first primer that described joint junction fragment is carried out the first amplification, it is thus achieved that the first amplified production；
(5) utilize the test kit described in any one of claim 1-3 that described first amplified production is captured, it is thus achieved that described mesh
Mark region；And,
(6) utilizing the second primer that described target area is carried out the second amplification, it is thus achieved that the second amplified production, described second amplification is produced
Thing constitutes described target area sequencing library.
Method the most according to claim 5, it is characterised in that described first primer sequence such as SEQ ID NO:1 and SEQ
Shown in ID NO:2；Described second primer sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4.
Method the most according to claim 5, it is characterised in that described samples sources is in human or animal；Described target area
For Colon and rectum related gene region.
8. a sequence measurement, it is characterised in that including:
Method according to claim 5 builds target area sequencing library；
Checking order described target area sequencing library, it is thus achieved that sequencing data, described sequencing data is made up of multiple reading sections；Its
In, NextSeq CN500 carries out described order-checking.
9. the method detecting target area variation, it is characterised in that include,
(1) method utilizing claim 8, it is thus achieved that sequencing data, filters described sequencing data, and described filtration includes
Remove the uncertain base ratio reading section more than 10% and/or the ratio of base number that base mass value is not more than 5 is not less than
The reading section of 50%；
(2) described sequencing data and reference sequences are carried out the first comparison, it is thus achieved that the first comparison result, get rid of the first comparison knot
The reading section that two reading sections of a reading section centering in Guo are identical is right, and wherein said reference sequences is HG19；By described first ratio
A part for result Yu described reference sequences is carried out the second comparison, it is thus achieved that the second comparison result；Described comparison result is carried out
Refiltering, described filtration includes getting rid of in comparison base mismatch number and reads sections more than 3, it is thus achieved that SNP in described target area,
At least one in InDel, SV and CNV variation；Wherein, during a part for described reference sequences includes target area reference sequences
The mismatched regions that causes, each known InDel site.
10. the system detecting target area variation, it is characterised in that include,
Nucleic acid acquisition device, for obtaining the nucleic acid in sample to be tested, described nucleic acid is made up of multiple DNA fragmentations, described DNA sheet
Section is from the genomic DNA ruptured and/or free DNA fragmentation, and described DNA fragmentation has flat end；
Add base A device, be used for adding base " A " and hold to the 3 ' of described DNA fragmentation, it is thus achieved that there is the DNA fragmentation of sticky end A；
Joint connection, for jointing in the two ends of described sticky end fragment, it is thus achieved that joint junction fragment；
First amplification device, is used for utilizing the first primer that described joint junction fragment is carried out the first amplification, it is thus achieved that the first amplification
Product；
Acquisition equipment, for utilizing the test kit described in any one of claim 1-3 that described first amplified production is captured,
Obtain described target area；And,
Second amplification device, is used for utilizing the second primer that described target area is carried out the second amplification, it is thus achieved that the second amplified production；
Sequencing device, for checking order described amplified production, it is thus achieved that described target area variant sites information, variation includes
At least one in SNP, InDel, SV and CNV variation.
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| CN113186262A (en) * | 2021-04-09 | 2021-07-30 | 上海锐翌生物科技有限公司 | Method and kit for rapid quantification of MGI platform high-throughput sequencing library |
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| CN113278611A (en) * | 2021-03-07 | 2021-08-20 | 华中科技大学同济医学院附属协和医院 | Capture sequencing probes and uses thereof |
| CN113186262A (en) * | 2021-04-09 | 2021-07-30 | 上海锐翌生物科技有限公司 | Method and kit for rapid quantification of MGI platform high-throughput sequencing library |
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