CN109280702A - Determine the method and system of individual chromosome textural anomaly - Google Patents

Determine the method and system of individual chromosome textural anomaly Download PDF

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CN109280702A
CN109280702A CN201710602834.3A CN201710602834A CN109280702A CN 109280702 A CN109280702 A CN 109280702A CN 201710602834 A CN201710602834 A CN 201710602834A CN 109280702 A CN109280702 A CN 109280702A
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candidates
results set
full
exception type
type
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徐凤萍
王文婧
叶玲飞
杨振军
袁剑颖
徐金金
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BGI Shenzhen Co Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention proposes a kind of methods of determining individual chromosome textural anomaly.This method comprises: (1) utilizes at least one of karyotyping and microarray chip analysis, the first candidates textural anomaly type of the individual is determined, to obtain the first candidates Exception Type set；(2) genome sequencing is carried out to the tissue samples of the individual, and the first data analysis is carried out to obtained sequencing result, to obtain the second candidates Exception Type set；(3) genome sequencing is carried out to the unicellular sample of the individual, and the second data analysis is carried out to obtained sequencing result, to obtain third candidates Exception Type set；(4) it is based on the first candidates Exception Type set, the second candidates Exception Type set and third candidates Exception Type set, determines the final chromosomal structural abnormality type of the individual.

Description

Determine the method and system of individual chromosome textural anomaly
Technical field
The present invention relates to field of biological detection, in particular it relates to the method for determining individual chromosome textural anomaly And system.
Background technique
Chromosomal variation plays vital effect in influencing human health, especially with intellectual development, cancer, no The diseases such as pregnant infertility have close association.Since the world chromosome of Paris in 1971 names meeting, it has been found that Ren Leiran More than colour solid numerical abnormality and structural aberration 3000 have confirmed that more than chromosomal disorder syndrome 100, feeblemindedness and growth at present Developmental lag is the common trait of chromosomal disorder.Down syndrome (Down ' s syndrome) is the most common chromosome of the mankind Disease, other known chromosome integrated disease include DiGeorge syndrome, Patau syndrome, Williams syndrome, Prader-Willi and Angelman syndrome etc..And the other kinds of structural aberration of chromosome such as transposition (translocation), the fragmentation (chromothripis) of inversion (inversion) even chromosome be also cause a disease it is lethal One of the major reasons.Talkowski team in 2012 reports an example fetus with CHARGE syndrome stillborn foetus, reason Lead to gene C HD7 Gene Fusion and functionally inactive for the spontaneous balanced translocation of No. 6 and No. 8 chromosomes.To preimplantation embryo, Pregnant woman carries out corresponding detection means can find to take corresponding intervening measure in turn in time, can be significantly reduced neonatal Birth defect rate improves the eugenic rate of population.On the other hand, the distortion of chromosome and the fusion formed by distortion It is one of the occurrence and development important mechanisms of cancer.Nineteen sixty, first fusion was just by Nowell and Hungerford etc. white It is found in the leucocyte of blood disease chronic granulocytic leukemia.And there are many fusions to be identified as causing a disease for tumour so far Mutation, as the TMRPSS2-ERG in prostate cancer reaches 50% in Caucasian.Therefore, pass through early stage noninvasive detection hand The such distortion of Duan Jianding will bring historical breakthrough for the early diagnosis of tumour and intervention.
On the other hand, for sieving and diagnosis before being implanted into and the diagnosis of pregnant woman's Prenatal Screening, the noninvasive dyeing of doubtful cancer patient Body Distortion Detect demand, there is no effective detection method at present.Currently, tested and analyzed after single celled extraction especially into Diagnosis and screening before the relatively broad implantation for being applied to tumor research and body early embryo of row high-flux sequence.Peason HA in discovery pregnant woman blood in 1967 there are fetal nucleated red blood (Fetal Nucleated Red Blood Cell, NRBC), and Thomas Ashworth has found that circulating tumor is thin inside the blood samples of patients in 1869 with metastatic cancer earlier Born of the same parents (Circulating Tumor cells, CTCs) etc., this non-invasive manner detection chromosomal structural aberration provide possibility.Separately Outside, lead to be implanted under powered distortion existing for the body early embryo of part and exist only in a few cell (such as early carcinoma is thin Born of the same parents) in mutation can also be restored by Single cell analysis technology.
It is complete early in Talkowski team in 2011 using high-flux sequence progress transposition recombination detection based on genomic DNA It searches, but and not yet in effect solves due to building library sequencing and to compare analysis bring Systematic Errors bring high the later period at effective False positive.
Therefore, the detection method of chromosomal structural abnormality is still needed further to develop and improve.
Summary of the invention
The application is that inventor is proposed based on the discovery to following problems and the fact:
Karyotyping is widely applied in clinical diagnosis at present as the goldstandard of detection chromosome aberration in the prior art, But because its precision is lower (exception for being only capable of detection 5Mb or more), and can not the variation of Accurate Diagnosis cryptostructural.Microarray Chip (array Comparative Genomic Hybridization, aCGH) is at present via American National food drug Supervision Bureau recommends to promote and apply in invasive pre-natal diagnosis, but its to be only capable of detecting when that dosage (dosage) increases or decreases abnormal Become type.And the high-flux sequence detection technique developed at present detects for unicellular chromosomal variation, is also only capable of examining Micrometer lacks micro- repetition, and for inversion, balanced translocation, there is presently no effective high-flux detection methods.
The present invention is directed to solve at least some of the technical problems in related technologies.Present invention group Team breaks through the limitation of traditional detection method, and the technology of a set of detection chromosomal structural variation is developed for unicellular sample, The technology plays a significant role in the solution chromosomal disorders screening, cancer parting and the medication guide the problems such as.And it invents Accuracy benefits of the people based on high throughput sequencing technologies are developed and are sequenced based on full-length genome low depth, built using big demographic data Method that is vertical to screen out false positive caused by systemic mistake referring to collection, being able to detect most of pathogenic chromosomal structural variation, The larger recall rate for improving newborn's defect.
In the first aspect of the present invention, the invention proposes a kind of methods of determining individual chromosome textural anomaly.According to The embodiment of the present invention, which comprises (1) utilize at least one of karyotyping and microarray chip analysis, determine institute The first candidates textural anomaly type of individual is stated, to obtain the first candidates Exception Type set, described the One candidates Exception Type set is as goldstandard results set；(2) full genome is carried out to the tissue samples of the individual Group sequencing, and the first data analysis is carried out to obtained sequencing result, to obtain the second candidates Exception Type collection It closes, it is preferable that the tissue samples are blood sample；(3) genome sequencing is carried out to the unicellular sample of the individual, and Second data analysis is carried out to obtained sequencing result, to obtain third candidates Exception Type set, it is preferable that The cell sample is lymphocyte sample；(4) based on the first candidates Exception Type set, the second candidate dyeing Body Exception Type set and third candidates Exception Type set determine the final chromosomal structural abnormality class of the individual Type.Cryptostructural is effectively realized using the method for determining individual chromosome textural anomaly according to an embodiment of the present invention The detection of variation, and effectively realize the efficient detection for unicellular chromosomal variation.
In the second aspect of the present invention, the invention proposes a kind of systems of determining individual chromosome textural anomaly.According to The embodiment of the present invention, the system comprises: goldstandard detection unit, the goldstandard detection unit are used to utilize karyotyping At least one of with microarray chip analysis, the first candidates textural anomaly type of the individual is determined, to obtain First candidates Exception Type set, the first candidates Exception Type set is as goldstandard results set； Tissue samples genome sequencing unit, the gene order-checking unit are used to carry out full genome to the tissue samples of the individual Group sequencing, and the first data analysis is carried out to obtained sequencing result, to obtain the second candidates Exception Type collection It closes, it is preferable that the tissue samples are blood sample；Unicellular sample genome sequencing unit, the unicellular sample are complete Gene order-checking unit is used to carry out genome sequencing to the unicellular sample of the individual, and to obtained sequencing result The second data analysis is carried out, to obtain third candidates Exception Type set, it is preferable that the cell sample is lymph Cell sample；Chromosomal structural abnormality analytical unit, the chromosomal structural abnormality analytical unit are used to wait based on described first Chromosome abnormality type set, the second candidates Exception Type set and third candidates Exception Type set are selected, Determine the final chromosomal structural abnormality type of the individual.Utilize determining individual chromosome structure according to an embodiment of the present invention Abnormal system effectively realizes the detection of cryptostructural variation, and effectively realizes and become for unicellular chromosome Different efficient detection.
Detailed description of the invention
Fig. 1 is sample collection flow chart according to an embodiment of the present invention；
Fig. 2 is that flow chart is sequenced in library according to an embodiment of the present invention of building；
Fig. 3 is the structural schematic diagram of the system of determining individual chromosome textural anomaly according to an embodiment of the present invention；
Fig. 4 is the structural schematic diagram of chromosomal structural abnormality analytical unit according to an embodiment of the present invention；
Fig. 5 is the structural schematic diagram of chromosomal structural abnormality analytical unit according to an embodiment of the present invention；
Fig. 6 is the structural schematic diagram of chromosomal structural abnormality analytical unit according to an embodiment of the present invention；
Fig. 7 is the structural schematic diagram of chromosomal structural abnormality analytical unit according to an embodiment of the present invention；And
Fig. 8 is the structural schematic diagram of chromosomal structural abnormality analytical unit according to an embodiment of the present invention.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
The method for determining individual chromosome textural anomaly
In the first aspect of the present invention, the invention proposes a kind of methods of determining individual chromosome textural anomaly.According to The embodiment of the present invention, which comprises (1) utilize at least one of karyotyping and microarray chip analysis, determine institute The first candidates textural anomaly type of individual is stated, to obtain the first candidates Exception Type set, described the One candidates Exception Type set is as goldstandard results set；(2) full genome is carried out to the tissue samples of the individual Group sequencing, and the first data analysis is carried out to obtained sequencing result, to obtain the second candidates Exception Type collection It closes, it is preferable that the tissue samples are blood sample；(3) genome sequencing is carried out to the unicellular sample of the individual, and Second data analysis is carried out to obtained sequencing result, to obtain third candidates Exception Type set, it is preferable that The cell sample is lymphocyte sample；(4) based on the first candidates Exception Type set, the second candidate dyeing Body Exception Type set and third candidates Exception Type set determine the final chromosomal structural abnormality class of the individual Type.Cryptostructural is effectively realized using the method for determining individual chromosome textural anomaly according to an embodiment of the present invention The detection of variation, and effectively realize the efficient detection for unicellular chromosomal variation.
According to an embodiment of the invention, the chromosomal structural abnormality includes structure variation SV and copy number variation CNV At least one.Unicellular chromosomal structural variation can be effectively realized using detection method according to an embodiment of the present invention and/or is copied Shellfish number variation.It should be noted that the position of structure variation SV and copy number variation CNV are not particularly limited, structure variation SV The different location of chromosome can be both located at copy number variation CNV, may be alternatively located at the same position of chromosome, if being located at dyeing The same position of body then can both detect single structure variation SV or copy number variation CNV using method described herein, Structure variation SV can be detected and copy situation when number variation CNV is existed simultaneously under two kinds of variation cross influences.
According to a particular embodiment of the invention, step (4) further comprises: to the second candidates Exception Type Do not occur in the first candidates Exception Type set in set and the third candidates Exception Type set Candidates Exception Type is verified, and is excluded unverified candidates Exception Type, was passed through to obtain Second candidates Exception Type set of filter and the third candidates Exception Type set by filtering, the process Second candidates Exception Type set of filtering is as full-length genome results set, the third candidate dye by filtering Colour solid Exception Type set is as unicellular results set.
Another specific example according to the present invention, the verifying is by PCR breakpoint method, qPCR method and aCGH microarray core What at least one of piece method carried out.
Another specific example according to the present invention, step (4) further comprises: (a) being based on the full-length genome result set It closes and the unicellular results set, the determining candidates Exception Type existed only in the unicellular results set； (b) parallel laboratory test verifying is carried out to step (3) using check sample known to mutation type；And if (c) the parallel reality The result tested is not more than the mutation type of the check sample, then exists only in the unicellular results set described in reservation Candidates Exception Type, if the result of the parallel laboratory test is more than the mutation type of the check sample, from described Removal exists only in the candidates Exception Type in the unicellular results set in unicellular results set, to obtain By the unicellular results set of filtering.
Still another embodiment according to the present invention, step (4) further comprises: (i) is based on the full-length genome result Set and the unicellular results set determine the candidates exception class existed only in the full-length genome results set Type；(b) parallel laboratory test verifying is carried out to step (2) using check sample known to mutation type；And if (c) described parallel The result of experiment is not more than the mutation type of the check sample, then exists only in the full-length genome results set described in reservation In candidates Exception Type, if the result of the parallel laboratory test be more than the check sample mutation type, from Removal exists only in the candidates Exception Type in the full-length genome results set in the full-length genome results set, To obtain the full-length genome results set by filtering.
Still another embodiment according to the present invention, step (4) further comprises: (i) is based on the full-length genome result Set and the unicellular results set determine the candidates exception class existed only in the full-length genome results set Type；(ii) sequencing and analysis for re-starting step (2) to the tissue samples are to confirm that described exist only in the full base Because whether the candidates Exception Type in group results set repeats；And (iii) if it is described exist only in it is described Candidates Exception Type in full-length genome results set repeats, then exists only in the full-length genome described in reservation Candidates Exception Type in results set, if the candidate dye existed only in the full-length genome results set Colour solid Exception Type does not repeat, then exists only in the candidates in the full-length genome results set described in deletion Exception Type, to obtain the full-length genome results set by filtering.
According to a particular embodiment of the invention, in step (iii), if described exist only in the full-length genome result Candidates Exception Type in set does not repeat, and after the parameter of set-up procedure (2), repeats step (ii), such as The candidates Exception Type existed only in the full-length genome results set described in fruit does not repeat, then deletes institute State the candidates Exception Type existed only in the full-length genome results set.
Still another embodiment according to the present invention, step (4) further comprises: (A) is based on the full-length genome result Set and the unicellular results set determine the candidates exception class existed only in the full-length genome results set Type；(B) unicellular sample is resurveyed, and repeats step (3) and carries out parallel laboratory test；And (C) if the parallel laboratory test As a result the candidates Exception Type in the full-length genome results set is existed only in described in occurring in, then reservation is described only The candidates Exception Type being present in the full-length genome results set, if still without appearance in the parallel laboratory test The candidates Exception Type existed only in the full-length genome results set, then from the full-length genome result set Removal exists only in the candidates Exception Type in the full-length genome results set in conjunction, to obtain by filtering Full-length genome results set.
In order to make it easy to understand, inventor further illustrates determining individual chromosome textural anomaly with reference to Fig. 1 and Fig. 2 The process of method:
(1) sample collection
40-every carrier persons of chromosome abnormality carrier peripheral blood sample are collected to isolate from peripheral blood outside 2 All hemolymphs save peripheral blood and lymphocyte respectively.The above case all uses caryogram and the method for Array to carry out chromosome Abnormality detection, while (chromosome abnormality complexity judgment criteria is simple different to the chromosome abnormality complexity of determining case Normal: SV and CNV does not have intersection, and complicated abnormal: SV and CNV have intersection).
(2) library sequencing is built
(1) gDNA level high-flux sequence: human peripheral blood extracts gDNA, is carried out building library sequencing, benefit with existing conventional method Chromosomal structural variation situation analysis is carried out to the result after sequencing with existing information analysis process.Sequencing result is again and in (one) Caryogram and Array method detection chromosomal variation situation (goldstandard result) compare, all sequencing results are divided into Goldstandard reappears and non-goldstandard result.The sequencing result of non-goldstandard uses PCR breakpoint experimental verification SV, qPCR/aCGH again Experimental verification CNV retains and records experimental result, is the chromosome abnormality that genuine result adds to each case by experimental verification In information.
(2) full-length genome unicellular DNA level sequencing: is carried out using MDA method to separated peripheral blood lymphocytes Amplification, carries out WGA product to build library sequencing (seeing below detailed description) again, carries out chromosomal structural variation analysis to sequencing result.It is right In the experimental verification (later Unify legislation examine for WGA product) of WGA sequencing result, using PCR breakpoint experimental verification SV, The material of qPCR/aCGH experimental verification CNV, experimental verification are WGA product rather than gDNA.It will be in WGA product sequencing result and (2) The chromosome abnormality information of each case be compared, WGA sequencing result is divided into result and is reappeared, it is known that result is not reappeared, With additional detection result three classes.Known results are not reappeared and carry out the inspection of WGA product, experiment results are consistent, retain detection As a result, if inconsistent debug information analysis process.To additional detection as a result, first construct unicellular control collection into Row refilters, with removal system error etc., after using experimental verification and the debugging of process determine that the chromosome of necessary being becomes It is different.
(3) WGA product builds library and sequencing scheme: using Qiagen REPLI-g Single Cell Kit to unicellular sample Product carry out MDA amplification, and amplification condition is carried out referring to kit specification.After the completion of MDA, 25ugWGA product is taken, is used Hydroshear is interrupted, and recycles 3~8K segment, subsequent to build library using old process.Sequencing scheme: every 8 samples construct 1 text Library, upper 4 lane (30G data) carry out CG platform PE50+10 sequencing.
According to a particular embodiment of the invention, at least one of first data analysis and second data analysis be It carries out: (a) comparing the sequencing result and genome reference sequences through the following steps, the sequencing result is by multipair reading Length is to composition；(b) it is based on the comparison result of step (a), determines that described multipair every a pair for reading long pair is referred in the genome Physical distance in sequence；(c) based on the physical distance obtained in step (b), the multipair reading length is positive to differentiation Often matching set and abnormal matching set；(d) the reading length in the abnormal matching set is clustered, to obtain multipair reading Long cluster pair；And the breakpoint and chromosome abnormality type of chromosomal structural abnormality (e) are determined based on the multipair long cluster pair of reading.
According to the specific example of invention, in step (c), by by the physical distance and scheduled nucleic acid fragment length It compares, the multipair reading length is gathered and exception matching set normal matching is divided into, wherein the scheduled nucleic acid is long Degree is the Insert Fragment size based on the genome sequencing and determination.
According to the specific example of invention, after step (d), before step (e), further comprise: being grown based on each reading Long compactness and linear dependence are read contained in cluster pair, multipair read long cluster to being filtered to described.
Specific example according to the present invention, the compactness are respectively to read the long and cluster based on the long cluster centering of reading What the distance between center variance determined.
Specific example according to the present invention, the compactness are determined based on following equation:
Wherein, x1,x2,x3…xnIndicate the position of the reads of the long cluster of reading, n indicates to read strip number, and M is locative average Value, s2Indicate variance, wherein the position of reads refers to the distance counted from the 5 ' ends of read, the calculating of the average value M of position Formula is M=x1+(xn-x1)/2。
The long cluster centering of reading is located at by specific example according to the present invention in advance before calculating the compactness Described long cluster at least part long to the reading within length range both ends 25% of reading excludes.As within 22%, 20% with It is interior, within 15%, within 10%, within 5%, within 1%.
Specific example according to the present invention, the linear dependence are by reading long cluster to the reading progress for being included to described Row t-test correlation test and determination.
Specific example according to the present invention further comprises in step (e): (e-1) is based on the long cluster centering institute of reading The long matching relationship with the genome reference sequences of the reading contained, determines the type of the structure variation；(e-2) based on described It reads reading contained in long cluster pair and grows the matching position on the genome reference sequences, determine the breakpoint of the structure variation Range.
Specific example according to the present invention, in step (e-2), for variation is repeated, selection reads long cluster described in pairs Range between two farthest positions of the distance that is matched to in simultaneously extends preset distance outward, as the breakpoint model It encloses.
Specific example according to the present invention, in step (e-2), for deletion mutation, selection reads long cluster described in pairs Range between two nearest positions of the distance that is matched to in simultaneously extends preset distance inwardly, as the breakpoint model It encloses.
Specific example according to the present invention, in step (e-2), comprising: such long pair of a pair of of the reading of (e-2-1) selection, The reading is long to be present in described read in long cluster to only one reading length；(e-2--2) by the another of the selected the pair of long centering of reading One matching position for reading to grow on the genome reference sequences is as the break point range.
Specifically, at least one of the first data analysis and second data analysis are to carry out through the following steps :
(1) it is sequenced by CG platform, obtains the sequencing data of individual whole genome, the data are long to (reads by multipair reading Pair it) forms, the long sequence (reads) of every two readings, which partners, reads long pair.Reads pair is located at measured dyeing The both ends of body segment, and respectively from the normal chain and minus strand of corresponding chromosome, or it is simultaneously from corresponding chromosome just Chain and minus strand.Known to transposition between different chromosomes be divided between same arm and different arm (chromosome point is long-armed and short Arm), if it is the transposition between same arm, then one end is normal chain, one end is minus strand, but if it is the transposition between different arm, It so will be just or negative.
(2) sequencing data is compared with human genome reference sequences (hg19), is set according to the parameter for comparing software It sets, each pair of reads pair allows n mispairing (mismatch), can preferentially take 1 or 2, specifically can be according to data the case where It is adjusted, compares software and alignment parameters (presently mainly adjustment mispairing number, and the general feelings of mispairing number with no restrictions Condition can be arranged 1, for it is unicellular parameter can be adjusted so as to it is looser a bit, because of the unicellular quality of data compared to many cells It can be without so good).Comparison result will generate normal matching set and abnormal matching set.Normal matching set is reads Two reads in pair are matched to the identical chromosome of reference sequences, and positive minus strand positional relationship is consistent with reference sequences, Being coincide between reads pair by the physical distance of analytical calculation and the physical distance of reference sequences, (read pair comes from The nucleic acid fragment of one library molecule so its distance on sample is no more than the average length of sequencing library, and is referring to The physical distance of sequence, be read pair is matched with reference sequences positioning after the distance between, nucleic acid fragment is that have a model It encloses, this range can be understood as the physical distance of the reads of sample, and reads is compared to human genome and refers to sequence Also a physical distance can be obtained when in column, is exactly judged according to the two physical distances).Abnormal matching set and with The extremely matched reads pair of reference sequences, the set include in following kind of reads pair:a.reads pair Reads has been respectively matched to the different chromosomes of reference sequences, this kind of Reads and the related (balanced translocation of transposition textural anomaly With non-equilibrium transposition).Reads in b.reads pair has been matched to the same chromosome of reference sequences, but reads pair Between misfitted by the physical distance of analytical calculation and the physical distance of reference sequences, as inversion (occur inversion if, reads Between not only direction can change, their distance is also can be changed), missing is (if a pair of reads pair The distance between be less than human genome reference sequences on distance just illustrate that this is lacked between reads pair.It lacks The region of mistake is not no reads, but the distance between reads pair for being across this section of absent region is with human genome Distance on reference sequences is different) or repeat.
(3) reads in abnormal set of matches is clustered into cluster according to the position being matched to, contains in each cluster and comes from one group The long reads of single-ended reading of reads pair, the long sequence of the reading of the corresponding other end are located in another cluster；Cluster is obtained Cluster is filtered, including, calculate the compactness of each cluster, filter out compactness be unsatisfactory for preset requirement cluster and with Its pairs of cluster obtains the filtered result cluster that long pair is read containing the first kind, for judging that chromosome translocation structure is different Normal generation.Inversion is but the object between reads pair by comparing between detection reads pair to phase homologous chromosomes Reason distance and the physical distance for not meeting human genome reference sequences, and reads pair is contrary；And lack or Repetition is to arrive phase homologous chromosomes by comparing between detection reads pair, but reads pair is less than or greater than human gene The physical distance of group reference sequences, and the direction reads pair does not change.
(5) in order to exclude interference that may be present as much as possible, (such as sample contamination, compares mistake, noise at sequencing mistake Deng) need further to be filtered clustering cluster as unit of cluster, filter type has following several: a. (one) is compact according to cluster Degree: the compactness for calculating each cluster (is come to carry out quantification treatment to compactness with variance, calculates the position of each Read in cluster The variance with the center of cluster or center of gravity is set, the smaller then compactness of variance is higher.Preferably, in the compactness for calculating each cluster When, it can abandon being the long sequence of reading in 5% to 25% positioned at the length range at the both ends of cluster, preferably 20%, it is outer to reduce Enclose influence of the data to calculated result), filter out cluster and the cluster pairs of with it that compactness is unsatisfactory for preset requirement.Cluster it is tight Cause degree reflects the stability of read distribution, shows whether read concentrates in a lesser section, it is however generally that, very Real structure variation can be submerged among numerous " environmental noises ", but the influence of " environmental noise " to entire full-length genome is basic It is uniformly, so substantially even distribution of trend is presented in complete sequence (it is of course also possible to will receive such as GC (guanine Guanine and cytimidine Cytosine) content etc. influence), and the Read in the place that true structure variation occurs, cluster The trend of similar normal distribution, therefore compactness, such as variance, the difference feelings that can be well reflected between cluster would generally be presented Condition.b.Linear dependence according to pairs of cluster: it is discontented to filter out linear dependence for the linear dependence for two clusters being calculated as pair The pairs of cluster of the preset requirement of foot.Linear dependence more focuses on the consistency that reads is distributed in pairs of cluster, i.e. performance reads Whether the distribution trend at both ends is almost the same, therefore linear dependence can more reflect the distribution situation inside pairs of cluster.C. foundation The control collection of normal sample: pairs of cluster is compared with the preset control collection comprising multiple normal samples, filters out life The number of middle normal sample reaches the pairs of cluster of preset threshold value.D. according to other auxiliary parameters: alleged auxiliary parameter includes each Kind facilitates further confirmation, specification configuration Exception Type or the parameter for helping to understand the details of textural anomaly.Through The filtering in various degree is crossed, the cluster of removal 80% or so can be probably removed, and the cluster that ordinary circumstance only has units leaves Come.
(6) presence of the result cluster obtained after filtering reflects the chromosomal structural abnormality that may have occurred respective type, For the more detailed information about textural anomaly of acquisition, need further to carry out data analysis to the result cluster of acquisition, for Different types of result cluster, can be used following analysis mode: if two a. adjacent long sequences of reading are in respectively affiliated reads Position on the contrary, obtain the two read the range between the position that long sequences match arrives as breakpoint range (i.e. reads all across Breakpoint has been crossed, the left-right position just them being matched to for such reads is as the range of breakpoint).Such case is usual It is related with balanced translocation, the two sides of breakpoint are distributed in the reads in cluster.B. the distance being matched to is obtained in pairs of cluster Range between two farthest positions extends outwardly presetting length respectively as occurring duplicate range, and from two positions (the distance between read pair variation is also to have, if the segment repetition occurred is bigger, between read pair Distance change also can be bigger, not only to examine reads number here, also to examine the distance inside reads pair, extend outwardly Presetting length, which refers to, can control the length for extending one times).C. two of the distance being matched to recently are obtained in pairs of cluster Range between position extends presetting length (i.e. for hair as the range lacked, and from two positions respectively inwards The region of raw missing, positional relationship of their cluster on reference genome are greater than actual distance in the sample, and Corresponding region does not have that the number of reads covering or reads are less with respect to the region on side, extends one times of length).
(7) breakpoint assembling is carried out using the data that analysis is completed.After the range for primarily determining breakpoint, by this range Interior reads is individually extracted, and is then compared these reads to the corresponding region of human genome reference sequences, is The range of breakpoint is further reduced, breakpoint assembling can also be carried out using the data not compared, for example, disconnected determined by obtaining Single-ended reads () around point range in setting range (such as 0.5~2 times of Insert Fragment size), from the data not compared All patch sequences are cut into N sections as patch sequence by pairs of reads therewith for middle extraction, and N is preferably 2, and by patch sequence The subsequence obtained after truncation is compared with reference sequences again, according to can normal matched result breakpoint region is carried out Assembling (i.e. if a pair of read pair only has in read comparisons, other one does not have in comparison, this not than Upper read is likely to just fall on breakpoint, the reads of such case is put together and is taken away with human genome with reference to sequence Column are compared the position that can be obtained by breakpoint and (since sequencing itself has certain error rate, finally or use PCR+ Sanger carries out last breakpoint verifying as goldstandard)).
(8) after completing assembling.The range of breakpoint can be effectively reduced, it on this basis, can be further according to locating for breakpoint Position range prepare probe, using other rice genome sequence means, such as Sanger sequencing etc., finally obtain accurate breakpoint Position, in order to which (i.e. qPCR is the experimental method verified to CNV to further progress for the research of breakpoint.And for disconnected Point verifying, is the design primer sequence near breakpoint first, then carries out PCR amplification to corresponding sample, later produces amplification Object carries out sanger sequencing, and then obtains the specific DNA sequence dna of PCR product, finally refers to sequence alignment to human genome In sequence, the position of breakpoint can be obtained.(goldstandard that PCR+Sanger sequencing at present is experimental verification)).
The system for determining individual chromosome textural anomaly
In the second aspect of the present invention, the invention proposes a kind of systems of determining individual chromosome textural anomaly.According to The embodiment of the present invention, with reference to Fig. 3, the system comprises: goldstandard detection unit 100, the goldstandard detection unit 100 are used In utilizing at least one of karyotyping and microarray chip analysis, the first candidates textural anomaly of the individual is determined Type, to obtain the first candidates Exception Type set, the first candidates Exception Type set is as gold Standard results set；Tissue samples genome sequencing unit 200, the gene order-checking unit 200 are used for the individual Tissue samples carry out genome sequencing, and to obtained sequencing result carry out the first data analysis, to obtain second Candidates Exception Type set, it is preferable that the tissue samples are blood sample；Unicellular sample genome sequencing list Member 300, the unicellular sample genome sequencing unit 300 are used to carry out full-length genome to the unicellular sample of the individual Sequencing, and the second data analysis is carried out to obtained sequencing result, to obtain third candidates Exception Type set, Preferably, the cell sample is lymphocyte sample；Chromosomal structural abnormality analytical unit 400, the chromosome structure are different Normal analytical unit 400 is used to be based on the first candidates Exception Type set, the second candidates Exception Type collection Conjunction and third candidates Exception Type set, determine the final chromosomal structural abnormality type of the individual, specifically, institute Stating chromosomal structural abnormality may include at least one of structure variation SV and copy number variation CNV.Using implementing according to the present invention The system of the determination individual chromosome textural anomaly of example effectively realizes the detection of cryptostructural variation, and effectively real The efficient detection for unicellular chromosomal variation is showed.
Preferably, the tissue samples genome sequencing unit and unicellular sample genome sequencing unit are suitable for logical It crosses the following steps and carries out at least one of the analysis of the first data and second data analysis:
(a) sequencing result and genome reference sequences are compared, the sequencing result is long to constituting by multipair reading；
(b) it is based on the comparison result of step (a), determines described multipair every a pair for reading long pair in the genome with reference to sequence Physical distance on column；
(c) based on the middle physical distance obtained of step (b), the multipair reading length is gathered normal matching is divided into With abnormal matching set；
(d) the reading length in the abnormal matching set is clustered, to obtain the multipair long cluster pair of reading；And
(e) based on the multipair long cluster pair of reading, the breakpoint and chromosome abnormality type of chromosomal structural abnormality are determined；
Optionally, in step (c), by the way that the physical distance compares with scheduled nucleic acid fragment length, by institute It states multipair reading length and gathers and exception matching set normal matching is divided into, wherein the scheduled length nucleic acid is to be based on institute State the Insert Fragment size of genome sequencing and determination；
Optionally, after step (d), before step (e), further comprise: being read contained in long cluster pair based on each Read long compactness and linear dependence, to it is described it is multipair read long cluster to being filtered,
Optionally, the compactness is based on the distance between the center read long cluster centering and respectively read length with the cluster What variance determined,
Optionally, the compactness is determined based on following equation:
Wherein, x1,x2,x3…xnIndicate the position of the reads of the long cluster of reading, n indicates to read strip number, and M is locative average Value, s2Indicate variance, wherein the position of reads refers to the distance counted from the 5 ' ends of read, the calculating of the average value M of position Formula is M=x1+(xn-x1)/2
Optionally, before calculating the compactness, long cluster centering is read positioned at the long cluster of reading to length by described in advance The long at least part of the reading spent within range both ends 25% excludes,
Optionally, the linear dependence is by reading long cluster to the long progress t-test correlation of the reading for being included to described Examine and determine,
Optionally, further comprise in step (e):
(e-1) it reads to read the long matching relationship with the genome reference sequences contained in long cluster pair based on described, really The type of the fixed structure variation；
(e-2) it reads to read to grow the matching position on the genome reference sequences contained in long cluster pair based on described, Determine the break point range of the structure variation,
Optionally, in step (e-2),
For variation is repeated, select between two farthest positions of the distance for reading to be matched in long cluster pair in pairs Range simultaneously extends preset distance outward, as the break point range,
Optionally, in step (e-2),
For deletion mutation, select between two nearest positions of the distance for reading to be matched in long cluster pair in pairs Range simultaneously extends preset distance inwardly, as the break point range,
Optionally, in step (e-2), comprising:
(e-2-1) a pair of as selection to read long pair, the reading is long to be present in described read in long cluster to only one reading length；
(e-2--2) selected the pair of another reading for reading long centering is grown on the genome reference sequences Matching position as the break point range.
Specifically, with reference to Fig. 4, the chromosomal structural abnormality analytical unit 400 further comprises: candidates are abnormal Type filter assemblies 410, the candidates Exception Type filter assemblies 410 are for described to second candidates Not in the first candidates Exception Type set in Exception Type set and the third candidates Exception Type set The candidates Exception Type of middle appearance is verified, and unverified candidates Exception Type is excluded, to obtain The second candidates Exception Type set by filtering and the third candidates Exception Type set by filtering are obtained, The second candidates Exception Type set by filtering is used as full-length genome results set, the by filtering Three candidates Exception Type set are as unicellular results set.Optionally, it is described verifying be by PCR breakpoint method, What at least one of qPCR method and aCGH micro-array chip method carried out.
Specifically, with reference to Fig. 5, the chromosomal structural abnormality analytical unit 400 further comprises: unicellular results set Filter assemblies 420, the unicellular results set filter assemblies 420 are used for: (a) being based on the full-length genome results set and institute Unicellular results set is stated, determines the candidates Exception Type existed only in the unicellular results set；(b) it uses Check sample known to mutation type enters the unicellular sample genome sequencing unit, carries out parallel laboratory test verifying；With And if (c) result of the parallel laboratory test is not more than the mutation type of the check sample, exists only in institute described in reservation The candidates Exception Type in unicellular results set is stated, if the result of the parallel laboratory test is more than the check sample Mutation type, then the candidate dyeing in the unicellular results set is existed only in from removal in the unicellular results set Body Exception Type, to obtain the unicellular results set by filtering.
Specifically, with reference to Fig. 6, the chromosomal structural abnormality analytical unit 400 further comprises: full-length genome result set Close the first filter assemblies 430, first filter assemblies of full-length genome results set 430 are used for: (i) is based on the full-length genome Results set and the unicellular results set determine that the candidates existed only in the full-length genome results set are different Normal type；(b) enter the tissue samples genome sequencing unit using check sample known to mutation type to carry out in parallel Experimental verification；And if (c) result of the parallel laboratory test is not more than the mutation type of the check sample, retain described in The candidates Exception Type in the full-length genome results set is existed only in, if the result of the parallel laboratory test is more than The mutation type of the check sample, then removal exists only in the full-length genome result from the full-length genome results set Candidates Exception Type in set, to obtain the full-length genome results set by filtering.
Specifically, with reference to Fig. 7, the chromosomal structural abnormality analytical unit 400 further comprises: full-length genome result set Close the second filter assemblies 440, second filter assemblies of full-length genome results set 440 are used for: (i) is based on the full-length genome Results set and the unicellular results set determine that the candidates existed only in the full-length genome results set are different Normal type；(ii) tissue samples genome sequencing unit is reentered to the tissue samples to be sequenced and analyzed, so as to Whether the candidates Exception Type existed only in the full-length genome results set described in confirmation repeats；And (iii) it if the candidates Exception Type existed only in the full-length genome results set repeats, protects Stay the candidates Exception Type existed only in the full-length genome results set, if it is described exist only in it is described Candidates Exception Type in full-length genome results set does not repeat, then exists only in the full base described in deletion Because of the candidates Exception Type in group results set, to obtain the full-length genome results set by filtering, optionally, In (iii), if the candidates Exception Type existed only in the full-length genome results set does not repeat Occur, after the parameter of adjustment genome sequencing unit, repeats (ii), if described exist only in the full-length genome result Candidates Exception Type in set does not repeat, then exists only in the full-length genome results set described in deletion In candidates Exception Type.
Specifically, with reference to Fig. 8, the chromosomal structural abnormality analytical unit 400 further comprises: full-length genome result set Close third filter assemblies 450, the full-length genome results set third filter assemblies 450 are used for: (A) is based on the full-length genome Results set and the unicellular results set determine that the candidates existed only in the full-length genome results set are different Normal type；(B) unicellular sample is resurveyed, and repeats to enter the progress of unicellular sample genome sequencing unit in parallel in fact It tests；And (C) if in the result of the parallel laboratory test occur described in exist only in the time in the full-length genome results set Chromosome abnormality type is selected, then exists only in the candidates exception class in the full-length genome results set described in reservation Type, if still without the candidates existed only in described in appearance in the full-length genome results set in the parallel laboratory test Exception Type then exists only in the candidate dye in the full-length genome results set from removal in the full-length genome results set Colour solid Exception Type, to obtain the full-length genome results set by filtering.
Embodiment
One, sample collection buys thousand human genomes from Coriell Institute for Medical Research Lymphoblastoid cell line.
Two, cell culture:
1) recovery of cell fast melt cell in 37 degree of water-baths, waits to take out when only remaining some solids and gently shake It shakes, makes thoroughly to thaw.It is transferred in 15mL centrifuge tube, isometric cell culture medium is added and is centrifuged, centrifugal condition are as follows: room Temperature, 300g, 5 minutes；Centrifuge model: Thermo scientific Sorvall ST 8.Discard supernatant.It is resuspended with culture medium Cell is transferred in culture dish.Normal culture.
2) cell culture
It is cultivated in 37 DEG C, 5%CO2 incubator with the culture medium that RPMI1640 plus 10% fetal calf serum are prepared, until thin Cell total amount reaches 1*10 after born of the same parents count5Quantity；
3) cell cryopreservation
The cell in 2) after culture is harvested to be suspended carefully after culture medium is removed in 300g centrifugation after five minutes with frozen stock solution at room temperature Born of the same parents, in packing to inward turning cryopreservation tube.Inward turning cryopreservation tube is put into program temperature reduction box, is placed in -80 DEG C and is at least no more than for 24 hours One week.Freeze formula of liquid :+10% dimethyl sulfoxide (DMSO) of+10% fetal calf serum of complete medium；
Three, are unicellular to be selected
The cell frozen first quick-thawing in 37 DEG C water baths before needing to select, after being diluted with PBS Individual cells are selected on Arcturus XT detection wind lidar instrument into 0.2ml EP pipe.
The unicellular DNA whole genome amplification of four,
1. genome amplification
A) consumptive material needed for testing Nuclease-free water etc. before amplification is put into super-clean bench, and ultraviolet irradiation 0.5~ 1h；
B) it takes equipped with single celled 200ul centrifuge tube, centrifuge gently gets rid of 5s；
C) Buffer D2 is prepared needed for taking, ready-to-use, system is as shown in table 1,
Table 1:
Ingredient Volume/ul
DTT(1M) 5
Reconstituted Buffer DLB 55
Total volume 60
D) 3.5ul Buffer D2 is added in singe-cell PCR tube wall, does not blow and beat, centrifuge gently gets rid of 5s；
E) 65 DEG C of incubation 10min (heat lid is set as 75 DEG C), are placed on ice at once after the reaction was completed；
F) 3.5ul Stop Solution is added along tube wall, does not blow and beat, centrifuge gently gets rid of 5s；
G) required preparation amplified reaction Master Mix is pressed, system is as shown in table 2:
Table 2:
Ingredient Volume/ul
Nuclease-free water 9
REPLI-gsingle cell reaction buffer 29
REPLI-gsingle cell DNA Polymerase 2
Total volume 40
H) every pipe is unicellular is added 40ul Master Mix along tube wall, flicks mixing, 300g centrifuge is got rid of at room temperature 10s is placed on ice；
I) PCR program is set, hot lid temperature 70 C:
Singe-cell PCR pipe is placed in PCR instrument by 30 DEG C of 65 DEG C of 8h, 12 DEG C of 10min hold, starts to react.
2. house-keeping gene detects MDA expanding effect
A) multi-PRC reaction (multiplex PCR), primer are configured to after working solution concentration (10uM) according to following ratio Primer Mix is prepared, system is as shown in table 3:
Table 3:
B) PCR system is prepared, system is as shown in table 4,
Table 4:
Ingredient Volume/ul
10xBuffer 3.0
dNTP(2.5mM) 3.2
PrimerMix(10uM) 3.0
100xBSA 0.2
rTaq(5U/ul) 0.4
NF-water 19.2
DNA template 1.0
Total 30
C) it after the completion of preparing, being expanded according to following conditions, condition is as shown in table 5,
Table 5:
D) after the completion of PCR, using 2% Ago-Gel, 10 μ l products, 120V electrophoresis 1h, ordinary circumstance electrophoresis detection: are taken Under, have in 8 genes 4 with positive for grade product.
The building of five, sequencing libraries and CG platform high-flux sequence
1) by DNA detection pass (i.e. have in 8 genes 4 with positive) carry out ultrasound afterwards and interrupt, be broken at random The large fragment of 3000-8000bp or so then adds connector A at the 5 ' of segment and 3 ' ends respectively, passes through the side PCR added with connector A Formula carries out fragment amplification.Amplified fragments are purified by Dynabeads M-280 Streptavidin MagneSphere, and segment after purification is used Ecop15 enzyme carries out digestion to DNA fragmentation, and recycles the segment after digestion with magnetic bead.It is similar with the connection of connector A, it is purifying The end of segment 5 ' and 3 ' after the recovery adds connector B respectively, is used for single-stranded cyclisation.The single-stranded loop ultimately generated is exactly flat in CG sequencing Platform carries out the library of machine sequencing.Finally we carry out high-flux sequence to each library to ensure that each sample meets sequencing Depth requirements.The average sequencing depth of each sample is 2X.
2) CG platform is anchored sequencing mode using joint probe, and the final product DNA single-stranded loop of library construction passes through The DNA nanosphere (DNB:DNA Nanobal) obtained after rolling-circle replication, DNB pass through the DNA nano-array of filling high density degree Probe anchoring sequencing approach is closed to identify sequence positioning, carry out unknown nucleotide sequence analysis in conjunction with a variety of probes.Existed by the way that fluorescence is imaged The step of each connection, we can determine whether the nucleotide sequences of each DNB.
3) after carrying out base calling, a large amount of reads sequence has been obtained, has then used Complete The comparison tool developed inside Genomics is initially compared；Based on initially comparing as a result, software will identify that and refer to Genome may differentiated region.
The analysis of six, data
With specific reference to the thirdly information analysis in technical solution of the present invention.
Seven, results are shown and recall rate is as shown in table 5
Table 5:
The primer that eight, verification results use is as shown in table 6
Table 6:
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.
SEQUENCE LISTING
<110>BGI-Shenzhen
<120>method and system of individual chromosome textural anomaly is determined
<130> PIDC3170477
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gcaactgaca gcaacgattt ct 22
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<212> DNA
<213> Artificial
<220>
<223>chr17-64-F-chr16-75-F reverse primer
<400> 30
aagtaaggag tgttgggagc agt 23
<210> 31
<211> 19
<212> DNA
<213> Artificial
<220>
<223>chr16-75-F-chr17-64-F forward primer
<400> 31
ggcagatgag aagggctga 19
<210> 32
<211> 23
<212> DNA
<213> Artificial
<220>
<223>chr16-75-F-chr17-64-F reverse primer
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atgttgctcg ttaatactcc ctg 23

Claims (21)

1. a kind of method of determining individual chromosome textural anomaly characterized by comprising
(1) at least one of karyotyping and microarray chip analysis are utilized, determines the first candidates knot of the individual Structure Exception Type, to obtain the first candidates Exception Type set, the first candidates Exception Type set As goldstandard results set；
(2) genome sequencing is carried out to the tissue samples of the individual, and the first data is carried out to obtained sequencing result Analysis, to obtain the second candidates Exception Type set, it is preferable that the tissue samples are blood sample；
(3) genome sequencing is carried out to the unicellular sample of the individual, and the second number is carried out to obtained sequencing result According to analysis, to obtain third candidates Exception Type set, it is preferable that the cell sample is lymphocyte sample；
(4) it is waited based on the first candidates Exception Type set, the second candidates Exception Type set and third Chromosome abnormality type set is selected, determines the final chromosomal structural abnormality type of the individual.
2. the method according to claim 1, wherein the chromosomal structural abnormality includes structure variation SV and copies At least one of shellfish number variation CNV.
3. the method according to claim 1, wherein what first data analysis and second data were analyzed At least one carry out through the following steps:
(a) sequencing result and genome reference sequences are compared, the sequencing result is long to constituting by multipair reading；
(b) it is based on the comparison result of step (a), determines described multipair every a pair for reading long pair on the genome reference sequences Physical distance；
(c) based on the physical distance obtained in step (b), by multipair readings it is long to divide into normal matching set with it is different Often matching set；
(d) the reading length in the abnormal matching set is clustered, to obtain the multipair long cluster pair of reading；
(e) read to read long compactness and linear dependence contained in long cluster pair based on each, to it is described it is multipair read long cluster into Row filtering；And
(f) based on the long cluster pair of multipair reading by filtering, the breakpoint and chromosome abnormality of chromosomal structural abnormality are determined Type.
4. according to the method described in claim 3, it is characterized in that, in step (c), by by the physical distance and predetermined Nucleic acid fragment length compare, it is the multipair reading is long to dividing into normal matching set and abnormal matching set, wherein institute Stating scheduled length nucleic acid is Insert Fragment size based on the genome sequencing and determination.
5. according to the method described in claim 3, it is characterized in that, the compactness is determined based on following equation:
Wherein, x1,x2,x3…xnIndicate the position of the reads of the long cluster of reading, n indicates to read strip number, the locative average value of M, s2 Indicate variance.
6. according to the method described in claim 5, it is characterized in that, before calculating the compactness, in advance by the reading Long cluster centering is located at described long cluster at least part long to the reading within length range both ends 25% of reading and excludes.
7. according to the method described in claim 3, it is characterized in that, the linear dependence is by reading long cluster to institute to described The reading that includes is long to carry out t-test correlation test and determination.
8. according to the method described in claim 3, it is characterized in that, further comprising in step (e):
(e-1) it reads to read the long matching relationship with the genome reference sequences contained in long cluster pair based on described, determines institute State the type of structure variation；
(e-2) it reads to read to grow the matching position on the genome reference sequences contained in long cluster pair based on described, determine The break point range of the structure variation.
9. according to the method described in claim 8, it is characterized in that, in step (e-2),
For variation is repeated, the range between two farthest positions of the distance for reading to be matched in long cluster pair in pairs is selected And extend preset distance outward, as the break point range.
10. according to the method described in claim 8, it is characterized in that, in step (e-2),
For deletion mutation, the range between two nearest positions of the distance for reading to be matched in long cluster pair in pairs is selected And extend preset distance inwardly, as the break point range.
11. according to the method described in claim 8, it is characterized in that, in step (e-2), comprising:
(e-2-1) a pair of as selection to read long pair, the reading is long to be present in described read in long cluster to only one reading length；
(e-2--2) on the genome reference sequences is grown into selected the pair of another reading for reading long centering With position as the break point range.
12. the method according to claim 1, wherein in step (4), comprising:
To in the second candidates Exception Type set and the third candidates Exception Type set not The candidates Exception Type occurred in one candidates Exception Type set is verified, and unverified time is excluded Chromosome abnormality type is selected, to obtain the second candidates Exception Type set by filtering and the third by filtering Candidates Exception Type set, the second candidates Exception Type set by filtering is as full-length genome knot Fruit set, the third candidates Exception Type set by filtering is as unicellular results set.
13. according to the method for claim 12, which is characterized in that it is described verifying be by PCR breakpoint method, qPCR method and What at least one of aCGH micro-array chip method carried out.
14. according to the method for claim 12, which is characterized in that step includes: in (4)
(a) it is based on the full-length genome results set and the unicellular results set, determination exists only in the unicellular knot Candidates Exception Type in fruit set；
(b) parallel laboratory test verifying is carried out to step (3) using check sample known to mutation type；And
If (c) result of the parallel laboratory test is not more than the mutation type of the check sample, existed only in described in reservation Candidates Exception Type in the unicellular results set, if the result of the parallel laboratory test is more than the control sample This mutation type then exists only in the candidate dye in the unicellular results set from removal in the unicellular results set Colour solid Exception Type, to obtain the unicellular results set by filtering.
15. according to the method for claim 12, which is characterized in that step includes: in (4)
(a) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(b) parallel laboratory test verifying is carried out to step (2) using check sample known to mutation type；And
If (c) result of the parallel laboratory test is not more than the mutation type of the check sample, existed only in described in reservation Candidates Exception Type in the full-length genome results set, if the result of the parallel laboratory test is more than the control The mutation type of sample then exists only in the full-length genome results set from removal in the full-length genome results set Candidates Exception Type, to obtain the full-length genome results set by filtering.
16. according to the method for claim 12, which is characterized in that step includes: in (4)
(i) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(ii) to the tissue samples re-start step (2) sequencing and analysis to confirm that it is described exist only in it is described complete Whether the candidates Exception Type in genome results set repeats；And
(iii) if the candidates Exception Type existed only in the full-length genome results set repeats, The candidates Exception Type in the full-length genome results set is existed only in described in then retaining,
If the candidates Exception Type existed only in the full-length genome results set does not repeat, The candidates Exception Type in the full-length genome results set is existed only in described in deletion, to obtain by filtering Full-length genome results set.
17. according to the method for claim 16, which is characterized in that in step (iii), if it is described exist only in it is described Candidates Exception Type in full-length genome results set does not repeat, and after the parameter of set-up procedure (2), repeats Step (ii), if the candidates Exception Type existed only in the full-length genome results set does not repeat It is existing, then the candidates Exception Type in the full-length genome results set is existed only in described in deletion.
18. according to the method for claim 12, which is characterized in that step includes: in (4)
(A) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(B) unicellular sample is resurveyed, and repeats step (3) and carries out parallel laboratory test；And
(C) if existing only in the candidate dye in the full-length genome results set described in occurring in the result of the parallel laboratory test Colour solid Exception Type then exists only in the candidates Exception Type in the full-length genome results set described in reservation, such as It is abnormal still without the candidates existed only in described in appearance in the full-length genome results set in parallel laboratory test described in fruit Type then exists only in the candidates in the full-length genome results set from removal in the full-length genome results set Exception Type, to obtain the full-length genome results set by filtering.
19. a kind of system of determining individual chromosome textural anomaly characterized by comprising
Goldstandard detection unit, the goldstandard detection unit be used for using karyotyping and microarray chip analysis at least it One, the first candidates textural anomaly type of the individual is determined, to obtain the first candidates Exception Type collection It closes, the first candidates Exception Type set is as goldstandard results set；
Tissue samples genome sequencing unit, the gene order-checking unit are used to carry out the tissue samples of the individual complete Gene order-checking, and the first data analysis is carried out to obtained sequencing result, to obtain the second candidates exception class Type set, it is preferable that the tissue samples are blood sample；
Unicellular sample genome sequencing unit, the unicellular sample genome sequencing unit are used for the individual Unicellular sample carries out genome sequencing, and carries out the second data analysis to obtained sequencing result, to obtain third Candidates Exception Type set, it is preferable that the cell sample is lymphocyte sample；
Chromosomal structural abnormality analytical unit, the chromosomal structural abnormality analytical unit are used for based on the described first candidate dyeing Body Exception Type set, the second candidates Exception Type set and third candidates Exception Type set, determine institute State the final chromosomal structural abnormality type of individual.
20. system according to claim 19, which is characterized in that the chromosomal structural abnormality include structure variation SV and At least one of number variation CNV is copied,
Preferably, under the tissue samples genome sequencing unit and unicellular sample genome sequencing unit are suitable for passing through Column step carries out at least one of the analysis of the first data and second data analysis:
(a) sequencing result and genome reference sequences are compared, the sequencing result is long to constituting by multipair reading；
(b) it is based on the comparison result of step (a), determines described multipair every a pair for reading long pair on the genome reference sequences Physical distance；
(c) based on the physical distance obtained in step (b), by multipair readings it is long to divide into normal matching set with it is different Often matching set；
(d) the reading length in the abnormal matching set is clustered, to obtain the multipair long cluster pair of reading；And
(e) based on the multipair long cluster pair of reading, the breakpoint and chromosome abnormality type of chromosomal structural abnormality are determined；
It optionally,, will be described more by the way that the physical distance compares with scheduled nucleic acid fragment length in step (c) Set is matched to normally matching set and exception is divided into length is read, wherein the scheduled length nucleic acid is based on described complete The Insert Fragment size of gene order-checking and determination；
Optionally, after step (d), before step (e), further comprise: reading to read length contained in long cluster pair based on each Compactness and linear dependence, to it is described it is multipair read long cluster to being filtered,
Optionally, the compactness is respectively to read the distance between long and the cluster center variance based on the long cluster centering of reading Determining,
Optionally, the compactness is determined based on following equation:
Wherein, x1,x2,x3…xnIndicate the position of the reads of the long cluster of reading, n indicates to read strip number, the locative average value of M, s2 Indicate variance,
Optionally, before calculating the compactness, long cluster centering is read positioned at the long cluster of reading to length model by described in advance The long at least part of the reading within both ends 25% is enclosed to exclude,
Optionally, the linear dependence is by reading long cluster to the long progress t-test correlation test of the reading for being included to described And determine,
Optionally, further comprise in step (e):
(e-1) it reads to read the long matching relationship with the genome reference sequences contained in long cluster pair based on described, determines institute State the type of structure variation；
(e-2) it reads to read to grow the matching position on the genome reference sequences contained in long cluster pair based on described, determine The break point range of the structure variation,
Optionally, in step (e-2),
For variation is repeated, the range between two farthest positions of the distance for reading to be matched in long cluster pair in pairs is selected And extend preset distance outward, as the break point range,
Optionally, in step (e-2),
For deletion mutation, the range between two nearest positions of the distance for reading to be matched in long cluster pair in pairs is selected And extend preset distance inwardly, as the break point range,
Optionally, in step (e-2), comprising:
(e-2-1) a pair of as selection to read long pair, the reading is long to be present in described read in long cluster to only one reading length；
(e-2--2) on the genome reference sequences is grown into selected the pair of another reading for reading long centering With position as the break point range.
21. system according to claim 19, which is characterized in that the chromosomal structural abnormality analytical unit further wraps It includes:
Candidates Exception Type filter assemblies, the candidates Exception Type filter assemblies are for described to described the Not in the first candidates in two candidates Exception Type set and the third candidates Exception Type set The candidates Exception Type occurred in Exception Type set is verified, and it is abnormal to exclude unverified candidates Type, it is different to obtain the second candidates Exception Type set by filtering and the third candidates by filtering Normal type set, the second candidates Exception Type set by filtering are described as full-length genome results set Third candidates Exception Type set by filtering as unicellular results set,
Optionally, the verifying is carried out by least one of PCR breakpoint method, qPCR method and aCGH micro-array chip method.
Optionally, the chromosomal structural abnormality analytical unit further comprises: unicellular results set filter assemblies, the list Cell results set filter assemblies are used for:
(a) it is based on the full-length genome results set and the unicellular results set, determination exists only in the unicellular knot Candidates Exception Type in fruit set；
(b) the unicellular sample genome sequencing unit is entered using check sample known to mutation type, carried out parallel Experimental verification；And
If (c) result of the parallel laboratory test is not more than the mutation type of the check sample, existed only in described in reservation Candidates Exception Type in the unicellular results set, if the result of the parallel laboratory test is more than the control sample This mutation type then exists only in the candidate dye in the unicellular results set from removal in the unicellular results set Colour solid Exception Type, to obtain the unicellular results set by filtering；
Optionally, the chromosomal structural abnormality analytical unit further comprises: the first filter assemblies of full-length genome results set, First filter assemblies of full-length genome results set are used for:
(i) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(b) the tissue samples genome sequencing unit is entered using check sample known to mutation type and carries out parallel laboratory test Verifying；And
If (c) result of the parallel laboratory test is not more than the mutation type of the check sample, existed only in described in reservation Candidates Exception Type in the full-length genome results set, if the result of the parallel laboratory test is more than the control The mutation type of sample then exists only in the full-length genome results set from removal in the full-length genome results set Candidates Exception Type, to obtain the full-length genome results set by filtering；
Optionally, the chromosomal structural abnormality analytical unit further comprises: the second filter assemblies of full-length genome results set, Second filter assemblies of full-length genome results set are used for:
(i) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(ii) to the tissue samples reenter tissue samples genome sequencing unit be sequenced and analyzed to confirm that Whether the candidates Exception Type existed only in the full-length genome results set repeats；And
(iii) if the candidates Exception Type existed only in the full-length genome results set repeats, The candidates Exception Type in the full-length genome results set is existed only in described in then retaining,
If the candidates Exception Type existed only in the full-length genome results set does not repeat, The candidates Exception Type in the full-length genome results set is existed only in described in deletion, to obtain by filtering Full-length genome results set,
Optionally, in (iii), if the candidates exception class existed only in the full-length genome results set Type does not repeat, and after the parameter of adjustment genome sequencing unit, repeats (ii), if it is described exist only in it is described complete Candidates Exception Type in genome results set does not repeat, then exists only in the full genome described in deletion Candidates Exception Type in group results set,
Optionally, the chromosomal structural abnormality analytical unit further comprises: full-length genome results set third filter assemblies, The full-length genome results set third filter assemblies are used for:
(A) it is based on the full-length genome results set and the unicellular results set, determination exists only in the full-length genome Candidates Exception Type in results set；
(B) unicellular sample is resurveyed, and repeats to enter the progress parallel laboratory test of unicellular sample genome sequencing unit；With And
(C) if existing only in the candidate dye in the full-length genome results set described in occurring in the result of the parallel laboratory test Colour solid Exception Type then exists only in the candidates Exception Type in the full-length genome results set described in reservation, such as It is abnormal still without the candidates existed only in described in appearance in the full-length genome results set in parallel laboratory test described in fruit Type then exists only in the candidates in the full-length genome results set from removal in the full-length genome results set Exception Type, to obtain the full-length genome results set by filtering.
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