CN104093858A

CN104093858A - Method, system and computer readable medium for determining whether chromosome number variation exists in biological sample

Info

Publication number: CN104093858A
Application number: CN201280068310.6A
Authority: CN
Inventors: 谢伟伟; 李旭超; 陈芳; 蒋浩君; 汪建; 王俊; 杨焕明; 张秀清
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2012-11-13
Filing date: 2012-11-13
Publication date: 2014-10-08
Anticipated expiration: 2032-11-13
Also published as: HK1197084A1; WO2014075228A1; CN104093858B

Abstract

Provided are a method, a computer readable medium and a system for determining whether a chromosome number variation exists in a biological sample. Wherein, the method for determining whether a chromosome number variation exists in a biological sample comprises: sequencing genome DNA of the biological sample, so as to obtain multiple sequencing data; comparing the sequencing data with a reference genome sequence of an organism, so as to obtain a unique comparison sequencing data set comprising multiple unique comparison sequencing data; determining a number M of the unique comparison sequencing data contained in the unique comparison sequencing data set; determining a number N of unique comparison sequencing data from a preset chromosome in the unique comparison sequencing data set; according to formula ChrN%=N/M, determining a calculation ratio ChrN% of the preset chromosome; and according to the calculation ratio ChrN% of the preset chromosome, determining whether a chromosome number variation for the preset chromosome exists in the biological sample.

Description

Method, system and computer readable medium for determining whether chromosome number variation exists in biological sample

Determine the method for numerical abnormalities of chromosomes in biological specimen, system and computer-readable Jie's shield priority information

Without technical field

The present invention relates to biomedical sector, specifically, being related to hereditary variation examines field, more specifically, the present invention relates to method, system and the computer-readable medium for determining numerical abnormalities of chromosomes in biological specimen.Background technology

Spontaneous abortion is one of most common complication of clinical pregnancy, and incidence accounts for 10%-20%.Its cause of disease is broadly divided into embryo's factor, maternal factors and environmental factor.Wherein the inhereditary material of embryo is extremely main cause, it has been reported that numerical abnormalities of chromosomes has accounted for 50%-60% in early abortion embryo, detects the chromosome situation of aborted fetus, understands spontaneous abortion reason, has directive significance to next tire gestation.

Being usually used in the method for diagnosis at present has chromosome karyotype analysis, FISH (FISHs）, CGH (comparative genome hybridizations）, MLPA (multiplex ligation-dependent probe amplifications）, STR-PCR (STR combination polymerase chain reaction techniques）Deng.At present, chromosome karyotype analysis is still " goldstandard " of chromosomal disorders detection, the numerical abnormalities of chromosomes of the overwhelming majority can be detected, but the tissue samples of miscarriage, by sample is outmoded, easy pollution, in incubation easily by the polluting of Disease in Infants cell, interval between diagnosis is longer, chromosome morphology is poor etc., and factor is influenceed, often lead to cell culture failure or fail to pinpoint a disease in diagnosis, mistaken diagnosis.And clinical FISH diagnostic techniques is applied to recently, using chromosome probe, in situ hybridization is carried out to the chorionic villi do not cultivated, diagnose numerical abnormalities of chromosomes, quick and precisely, but it is domestic at present when carrying out cause of disease searching for miscarriage sample, chromosome 13,18,21, X and Y are largely only related to using FISH probe, whole chromosomes are not detected simultaneously.

Thus, current aborted fetus chromosome abnormality detection still has much room for improvement.The content of the invention

It is contemplated that at least solving one of technical problem present in prior art.

In the first aspect of the present invention, the present invention proposes a kind of method that can effectively determine numerical abnormalities of chromosomes in biological specimen.Embodiments in accordance with the present invention, this method includes：It is sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；The sequencing data is compared with the biological reference gene group sequence, to obtain Obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；Determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;Determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;Based on formula ChrN%=N/M, the calculating ratio ChrN% of the predetermined chromosome is determined;And the calculating ratio ChrN% based on the predetermined chromosome, determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.Due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize method according to embodiments of the present invention, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine that the biological specimen whether there is numerical abnormalities of chromosomes for the chromosome.

In the second aspect of the present invention, the present invention proposes a kind of computer-readable medium.Be stored with instruction on embodiments in accordance with the present invention, the computer-readable medium, and the instruction is suitable to be executed by processor the method to determine numerical abnormalities of chromosomes in biological specimen through the following steps：Obtain multiple sequencing datas of the genomic DNA of the biological specimen；The sequencing data is compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；Determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;Determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;Based on formula ChrN%=N/M, the calculating ratio ChrN% of the predetermined chromosome is determined;And the calculating ratio ChrN% based on the predetermined chromosome, determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.As previously described, due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the computer-readable medium, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine that the biological specimen whether there is numerical abnormalities of chromosomes for the chromosome.

In the third aspect of the present invention, the present invention proposes a kind of system for determining numerical abnormalities of chromosomes in biological specimen.Just blunt according to embodiments of the invention, the system includes：Sequencing device, the sequencing device is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；Comparison device, the comparison device is connected with the sequencing device, for the sequencing data to be compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；Analytical equipment, the analytical equipment is connected with the comparison device, and whether there is numerical abnormalities of chromosomes for determining that the biological specimen is directed to predetermined chromosome, wherein, the analytical equipment further comprises：First computing module, first computing module is used to determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;Second computing module, second computing module is used to determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;3rd computing module, the 3rd computing module is used to be based on formula ChrN%=N/M, determines the calculating ratio ChrN% of the predetermined chromosome;And judge module, the judge module be used for the calculating ratio ChrN% based on the predetermined chromosome, determine the biological specimen for the predetermined chromosome whether there is numerical abnormalities of chromosomes.As previously described, because in theory, navigating to certain Content of the sum of sequencing data on chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the system, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine that the biological specimen whether there is numerical abnormalities of chromosomes for the chromosome.

In the fourth aspect of the present invention, the present invention proposes the system for determining numerical abnormalities of chromosomes in biological specimen.Embodiments in accordance with the present invention, the system includes：Sequencing device, the sequencing device is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；And foregoing computer-readable medium.As previously described, due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the system, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine whether the biological specimen has numerical abnormalities of chromosomes for the chromosome.

The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become apparent from the description below, or be recognized by the practice of the present invention.Brief description of the drawings

The above-mentioned and/or additional aspect and advantage of the present invention will be apparent and be readily appreciated that from description of the accompanying drawings below to embodiment is combined, wherein：

Fig. 1 shows the schematic flow sheet for determining the method for numerical abnormalities of chromosomes in biological specimen；And Fig. 2 shows the structural representation for determining the system of numerical abnormalities of chromosomes in biological specimen.Detailed description of the Invention

Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein same or similar label represents same or similar element or the element with same or like function from beginning to end.The embodiments described below with reference to the accompanying drawings are exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.

It should be noted that term " first,, " and second, it is only used for describing purpose, and it is not intended that indicating or implying relative importance or the implicit quantity for indicating indicated technical characteristic.Thus, " first " is defined, one or more this feature can be expressed or be implicitly included to the feature of " second ".It is further, in the description of the invention, unless otherwise indicated, " multiple, it is meant that two or more.

Method for determining numerical abnormalities of chromosomes in biological specimen

In the first aspect of the present invention, the present invention proposes a kind of method for determining numerical abnormalities of chromosomes in biological specimen.With reference to Fig. 1, this method includes：

S100:Genomic DNA is sequenced

In this step, it is sequenced first against the genomic DNA for the biological specimen for needing to be detected, it is many to obtain Individual sequencing data.Embodiments in accordance with the present invention, it is possible to use the type for the biological specimen that method of the invention is detected is not particularly restricted.Embodiments in accordance with the present invention, the biological specimen that can be used for detection is abortion tissue, wherein, the preferred fresh fine hair of the fine hair of fetal tissue, more preferably fetal tissue.Because, abortion tissue mainly includes the palace of the Qing Dynasty tissue of fetal tissue and parent, and object to be detected is fetus, and the fine hair in fetal tissue is foetal DNA direct sources, its DNA is complete, quality is preferable, therefore, the fine hair of fetal tissue is the optimal biological specimen for being used to determine the method for numerical abnormalities of chromosomes in biological specimen of the application present invention.In addition, because the DNA of the fetal tissue of missed abortion is second-rate, and have source of parents pollution, the fresh fine hair of fetal tissue should be typically chosen as biological specimen.And then, it can effectively detect the numerical abnormalities of chromosomes of aborted fetus using the method for the present invention.

Embodiments in accordance with the present invention, further comprise from biological specimen extract genomic DNA the step of.Embodiments in accordance with the present invention, can use salting out method, cross the conventional DNA extraction method such as post method and SDS methods from biological specimen extraction genomic DNA, it is preferred to use column chromatography.Wherein, the principle of column chromatography is：Cell or tissue exposes a DNA molecular for dew after the effect of cell pyrolysis liquid and Proteinase K, when it passes through the ethanol on pellosil post that can be combined with electronegative DNA molecular, genomic DNA in system is by reversible adsorption, after removing the impurity such as protein, lipid through rinsing liquid cleaning, the genomic DNA (product description that concrete principle and method are DP316 with reference to the product description and Tiangen companies article No. that Qiagen companies article No. is 56304 obtained in cell or tissue is eluted with refined solution）.

Embodiments in accordance with the present invention, in order to which the genomic DNA obtained is sequenced, can at random be interrupted to it.Just blunt according to embodiments of the invention, processing is interrupted at random to be carried out by using at least one of digestion, atomization, ultrasound and HydroShear methods.Preferably, using HydroShear methods（When the solution containing DNA is by passage compared with small area, fluid accelerates, the power of generation makes DNA be broken suddenly, and flow velocity and channel sized determine the size of DNA fragmentation, concrete principle and method referring to Life Sciences Wiki companies HydroShear specifications）, DNA molecular is interrupted to compare a certain size fragment of concentration.Embodiments in accordance with the present invention, are distributed in the range of 200 ~ 300bp by the master tape interrupted at random, i.e., the length of preferred DNA fragmentation is 200 ~ 300bp.

Embodiments in accordance with the present invention, the type for the sequencing device that can be used is not particularly restricted.According to a particular embodiment of the invention, advantage and high flux property energy in view of instrument portability, sequencing is by being carried out selected from least one of Roche/454 GS Junior, Illumina/MiSeq and Life Tecnologies/Ion Torrent PGM.The characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, further improve the efficiency for determining numerical abnormalities of chromosomes.Type is sequenced can be (unidirectional for single-end）Sequencing or Pair-end are (two-way）Sequencing.In one embodiment of the invention, described sequence measurement is Illumina/MiSeq, and sequencing type is two-way sequencing, and the fragment that obtained result is 150bp sizes is sequenced（reads ).Thus, it is possible to further improve the efficiency of subsequent analysis.

Those skilled in the art can select appropriate sequencing library construction method according to the microarray dataset used, and cylinder says it, build the method for sequencing library and can include： First, sample of nucleic acid to be detected is carried out after fragment, to obtain DNA fragmentation；

After DNA fragmentation is obtained, flat end processing is carried out to DNA fragmentation and base Α, and jointing are added in end, to obtain the DNA fragmentation with joint；And

DNA with joint is expanded, amplified production i.e. sequencing library is obtained.

Embodiments in accordance with the present invention, can introduce sequence label during sequencing library is built in sequencing library

Index, for example, can introduce Index, or the introducing sequence label Index in amplification procedure within a fitting.Thus, it is possible to by using different sequence labels for different samples, multiple detection samples are sequenced so as to realize simultaneously.Embodiments in accordance with the present invention, the sequence label length that can be used is 4-12bp, thus without other functions of influence addition sequence label Index DNA molecular.

S200:Obtain unique comparison sequencing data collection

In this step, the reference gene group sequence of resulting sequencing data and detected living species is compared, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas.

Embodiments in accordance with the present invention, in the present invention, when being detected for aborted fetus sample, the reference gene group sequence of the mankind used is the human genome reference sequences of latest edition in human genomic sequence reference sequences resulting after masking repetitive sequence, such as ncbi database.In a particular embodiment of the present invention, reference gene group sequence is the human genome reference sequences of version 37 (NCBI Build 37) in NCBI databases.

Embodiments in accordance with the present invention, can carry out sequence alignment, such as short oligonucleotide analysis bag obtained by those skilled in the art by any alignment programs（Short Oligo nucleotide Analysis Package, SOAP) and BWA comparisons（Burrows- Wheeler Aligner) at least one of carry out, sequencing data is compared with reference gene group sequence, position of the sequencing data in reference gene group is obtained.The default parameters that carrying out sequence alignment can use program to provide is carried out, or parameter is selected as needed by those skilled in the art.In a particular embodiment of the present invention, the comparison software used is SOAP aligner/soap2.

Term used in herein " uniquely comparing sequencing data " refers to, when sequencing data and reference gene group sequence are compared, only have the sequence of unique positions in reference gene group sequence, represented with Unique reads.In an embodiment of the present invention, in order to avoid the interference of repetitive sequence, need to remove those tandem sequence repeats for being positioned in human genome reference sequences and the DNA sequence dna of swivel base repeatable position, the DNA sequence dna of genome unique positions can be navigated to by only counting those, i.e., unique to compare sequencing data.Each DNA sequence dna of the DNA molecular from abortion tissue after interrupting and being sequenced can be positioned at specific chromosome by unique sequencing data that compares.

S300:Determine the calculating ratio ChrN% of predetermined chromosome

After unique comparison sequencing data collection is obtained, the unique comparison sequencing data for being positioned at each chromosome can be calculated, and calculate it with uniquely comparing the calculating ratio ChrN% of sequencing data collection. Specifically, embodiments in accordance with the present invention, the step further comprises：First, it is determined that uniquely comparing the number M of unique comparison sequencing data included in sequencing data collection;It is next determined that uniquely comparing the number N that sequencing data concentrates unique comparison sequencing data from predetermined chromosome;And based on formula ChrN%=N/M, determine the calculating ratio ChrN% of the predetermined chromosome

Embodiments in accordance with the present invention, the predetermined chromosome detected can be all chromosomes of living species, can be all autosomes and sex chromosome specific to human sample's such as fetal abortion sample.For example, just blunt according to embodiments of the invention, the predetermined chromosome that can be detected is selected from people 21,13, No. 18, at least one of X and Y chromosome, thus, it is possible to detect most of diseases related to numerical abnormalities of chromosomes.

S400:Judge whether numerical abnormalities of chromosomes

In this step, after its calculating ratio is obtained for predetermined chromosome, can the calculating ratio ChrN% based on the predetermined chromosome, determine biological specimen for the predetermined chromosome whether there is numerical abnormalities of chromosomes.

Embodiments in accordance with the present invention, ChrN% is the number for the unique comparison sequencing data that will be obtained on predetermined chromosome divided by uniquely compares the numerical value obtained after the number of unique comparison sequencing data included in sequencing data collection is standardized, thus, the numerical value is only relevant with the size of specific chromosome, and it is unrelated with the amount of sequencing data, so as to be analyzed with 01^% 46 respective situations of chromosome of living species such as people.For example, No. 1 chromosome is maximum chromosome in human genome（About 247M), and No. 21 chromosomes are minimum chromosome in human genome（About 47M), therefore be a definite value in certain sequencing total amount lower aprons for the sequencing result obtained by the normal biological sample of people.Although under certain sequencing condition and experiment condition, ChrN% is not fully directly proportional to chromosome size, substantially definite value.Thus, calculating ratio ChrN% is the baseline values of progress chromosome analysis in the present invention.

Specifically, embodiments in accordance with the present invention, the calculating ratio ChrN% based on predetermined chromosome determines that biological specimen further comprises for the predetermined chromosome with the presence or absence of numerical abnormalities of chromosomes：

Determine the calculating ratio ChrN% of the predetermined chromosome and the characteristic rate mean_ChrN of the predetermined chromosome./ ratio, to obtain the relative copy rate CR_ChrN of the predetermined chromosome of this in biological specimen,

Wherein, if the relative copy rate CR_ChrN of predetermined chromosome exceeds the normal copy rate scope of the predetermined chromosome in the biological specimen, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

According to a particular embodiment of the invention, the characteristic rate 1^＆^01^% of the predetermined chromosome is determined by least 30 normal samples, and the normal copy rate scope of the predetermined chromosome is to be determined by calculating at least 30 normal samples for 99% confidential interval that the calculating ratio ChrN% of the predetermined chromosome is distributed.

Thus, embodiments in accordance with the present invention, specific chromosome quantitative can be determined with normal sample with the presence or absence of difference by calculating relative copy rate CR_ChrN method in biological specimen such as abortion tissue sample, wherein by positioned at the corresponding biological specimen of exceptional value beyond normal chromosomal copy rate scope, regard as its chromosome quantitative to have differences with normal sample, that is, contaminate Colour solid aneuploidy.Specifically, the specific chromosome mean_ChrN% of ChrN% I of relative copy rate CR_ChrN=specific chromosome of examined samples of the specific chromosome of examined samples.

Wherein, the relative copy rate CR_ChrN of the specific chromosome of examined samples is bigger than normal or less than normal, illustrates that chromosome quantitative deviates normal value in the examined samples, when beyond certain limit, it is believed that its chromosome quantitative has notable difference with normal sample, that is, there is numerical abnormalities of chromosomes.In the present invention, the normal sample refers to the sample in the absence of specific chromosome abnormality.In one embodiment of the invention, the normal sample refers to that abnormal sample is not present in specific chromosome G banding.In the present invention, the ChrN% average values (i.e. mean_ChrN%) of the specific chromosome from normal sample can be according to from for example, at least 10, for example, at least 20, for example, at least 30, for example, at least 50, the ChrN% of the chromosome of for example, at least 100 normal samples is determined.In the present invention, the specific chromosome copies rate scope from normal sample can be according to from for example, at least 10, for example, at least 20, for example, at least 30, for example, at least 50,99% confidential interval of the ChrN% distributions of the chromosome of for example, at least 100 normal samples is determined.Furthermore, it is necessary to which explanation, is to judge whether CR_ChrN is bigger than normal or less than normal by following method in the present invention：Calculate the data distribution for the CR values for obtaining every chromosome of normal sample with colony check sample, and (U-test two-sided tests of being tested to the CR values of sample to be tested）, when it significantly peels off, can judge that CR_ChrN is bigger than normal or less than normal according to actual conditions.And then, it is possible to determine that the sample to be tested numerical abnormalities of chromosomes.

In one embodiment of the invention, the number of the normal sample is 20 normal males and 10 normal females, number in order as 1,2...30, wherein 1 ~ 20 is male's normal sample, 21 ~ 30 be female normal sample, then the ChrN% average values (mean_ChrN) of normal sample are calculated as follows：

1 ³⁰

(wherein N is No. 1 ~ No. 22 autosomes to Mean C rN=- Y ChrN M, and M is 1 ~ No. 30 normal sample）） _ 30 i _

Mean_ChrX (male)=- ^ ChrX_M (M is 1 ~ No. 20 male's normal sample)

20 _m=i

Mean_ChrY (male)=mono- ^ ChrY_M (M is 1 ~ No. 20 male's normal sample)

20 _m=i

Mean_ChrX (women)=- ^ ChrX-M (M is 21 ~ No. 30 female normal samples)

10

Mean_ChrY (women)=- ^ ChrY-M (M is 21 ~ No. 30 female normal samples)

10

Wherein, it should be noted that, because there is substantial amounts of breach in the fluctuation of sequencing and the Y chromosome of reference gene group sequence, even causing normal women sample also to have a small number of DNA sequence dnas to compare onto Y chromosome, but compared with male, ChY ° of women/b male is much smaller, and the 01 bifurcation % of women is 0.004 or so in such as embodiment, and the 01 bifurcation % of male is 0.114 or so.

Further, the CR_ChrN of each examined samples is resulted in, wherein, calculation formula is respectively： CR C r = ^chrN/ _{l Τ}

- / mean_ nrN

CR ChrX (male)=C vX/

- 、 / mean_ChrX Male )

CR ChrY (male)=ChrY/

- / mean_ ChrY ( Male )

CR_ChrX (female!■ gives birth to) (Female)

CR_ChrY (female!■ gives birth to) tean-ChrY (Female) is because male in the chromosome of women than lacking item chromosome X, it the substitute is chromosome Y, and X chromosome total length about 155M, and chromosome Y is only 59M, therefore ChrX% the or ChrY % normal distribution sample sets of a set of different sexes are must be set up when being detected to sex chromosome, most accurate analysis result could be drawn to the situation of sex chromosome according to different sex normal distribution data.

In one embodiment of the invention, take 30 normal samples to carry out chromosome analysis, required according to the conspicuousness of normal distribution（For example 1%) calculate the confidential interval of normal distribution（99% CI, CRL-CRR), and the situation of the relation different from confidential interval of the value based on CR_ChrN treats sample this progress chromosome analysis, it is specific as follows：

When CR_ChrN value is located between C and 0^, then the possibility that the sample has 99% is contained in the colony of a normal distribution, that is, difference is not present in its chromosome quantitative of the sample detected for normal sample；

When CR_ChrN value is less than CR_LWhen, then possibility of the sample more than 99% is rejected in the colony of a normal distribution, as outliers, i.e.,：There is missing for normal sample in its chromosome quantitative of the sample detected；When CR_ChrN value is more than CR_RWhen, then possibility of the sample more than 99% is rejected in the colony of a normal distribution, as outliers, i.e.,：There is increase for normal sample in its chromosome quantitative of the sample detected.

So far, pass through embodiments of the invention, numerical abnormalities of chromosomes that can be effectively to biological specimen is analyzed, for example, the tissue samples of miscarriage can be analyzed using high throughput sequencing technologies, the method detected to analyze, diagnose miscarriage reason made a variation by abortion tissue legacy.This method flux is high, specificity is high, accuracy rate is high.Computer-readable medium

In the second aspect of the present invention, the present invention proposes a kind of computer-readable medium.Be stored with instruction on embodiments in accordance with the present invention, the computer-readable medium, and the instruction is suitable to be executed by processor the method to determine numerical abnormalities of chromosomes in biological specimen through the following steps：

First, multiple sequencing datas of the genomic DNA of the biological specimen are obtained. Next, the sequencing data is compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas.Embodiments in accordance with the present invention, the reference gene group sequence is the human genome reference sequences of version 37 in ncbi database.According to a particular embodiment of the invention, using at least one of SOAP and BWA, the sequencing data is compared with the biological reference gene group sequence.

Then, determine unique number M for comparing unique comparison sequencing data included in sequencing data collection, determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome, based on formula ChrN%=N/M, the calculating ratio ChrN% of the predetermined chromosome is determined.

Finally, the calculating ratio ChrN% based on the predetermined chromosome, determines that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.Embodiments in accordance with the present invention, based on the calculating ratio ChrN% of the predetermined chromosome, determine that the biological specimen further comprises for the predetermined chromosome with the presence or absence of numerical abnormalities of chromosomes：Determine the calculating ratio 01^% of the predetermined chromosome and the characteristic rate mean_ChrN of the predetermined chromosome./ ratio, to obtain the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen, wherein, if the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen exceeds the normal copy rate scope of the predetermined chromosome, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

Embodiments in accordance with the present invention, the characteristic rate of the predetermined chromosome！^＆^0^% is determined by least 30 normal samples.Embodiments in accordance with the present invention, the normal copy rate scope of the predetermined chromosome is to be determined by calculating at least 30 normal samples for 99% confidential interval that the calculating ratio ChrN% of the predetermined chromosome is distributed.

As previously described, due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the computer-readable medium, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine whether the biological specimen has numerical abnormalities of chromosomes for the chromosome.

It should be noted that above for the description that the method for numerical abnormalities of chromosomes in biological specimen is carried out is determined, being also applied for the computer-readable medium, will not be repeated here.The system for determining numerical abnormalities of chromosomes in biological specimen

In the third aspect of the present invention, the present invention proposes a kind of system for determining numerical abnormalities of chromosomes in biological specimen.Just blunt according to embodiments of the invention, reference picture 2, the system includes：Sequencing device 100, comparison device 200 and analytical equipment 300.Embodiments in accordance with the present invention, sequencing device 100 is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas, comparison device 200 is connected with sequencing device 100, for the sequencing data to be compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas, analytical equipment 300 is connected with comparison device 200, and for determining the biological specimen for predetermined dye Colour solid whether there is numerical abnormalities of chromosomes.Embodiments in accordance with the present invention, the reference gene group sequence is the human genome reference sequences of version 37 in ncbi database.According to a particular embodiment of the invention, using at least one of SOAP and BWA, the sequencing data is compared with the biological reference gene group sequence.

Embodiments in accordance with the present invention, analytical equipment 300 further comprises：First computing module 310, for determining unique number M for comparing unique comparison sequencing data included in sequencing data collection;Second computing module 320 is used to determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;3rd computing module 330 is used to be based on formula ChrN%=N/M, determines the special calculating ratio ChrN% of the predetermined chromosome;And judge module 340 is used for the calculating ratio ChrN% based on the predetermined chromosome, determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.

Embodiments in accordance with the present invention, judge module 340 is suitable to pass through following, the calculating ratio based on the predetermined chromosome

ChrN%, determines that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome：Determine the calculating ratio 01^% of the predetermined chromosome and the characteristic rate mean_ChrN of the predetermined chromosome./ ratio, to obtain the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen, wherein, if the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen exceeds the normal copy rate scope of the predetermined chromosome, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

As previously described, due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the system, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine whether the biological specimen has numerical abnormalities of chromosomes for the chromosome.

It should be noted that the function of foregoing comparison device and analytical equipment can be performed by foregoing computer-readable medium.Thus, in the fourth aspect of the present invention, the present invention has also been proposed a kind of system for determining numerical abnormalities of chromosomes in biological specimen.Embodiments in accordance with the present invention, the system includes：Sequencing device, the sequencing device is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；And foregoing computer-readable medium.As previously described, due in theory, content of the sum of the sequencing data navigated on certain chromosome to the length and chromosome of the chromosome in biological specimen is proportional, thus, utilize the system, by navigating to the number of the sequencing data on certain chromosome, it can effectively determine whether the biological specimen has numerical abnormalities of chromosomes for the chromosome.

It should be noted that above for for determining the description that the method for numerical abnormalities of chromosomes in biological specimen is carried out, the system for being also applied for numerical abnormalities of chromosomes in the determination biological specimen will not be repeated here. The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and it should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition in embodiment, according to the technology or condition described by document in the art（Write such as with reference to J. Pehanorm Brookers, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press）Or carried out according to product description.Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional products that can be obtained by market.It is the producer article No of each reagent or kit in following bracket.The joint and sequence label of used sequencing derive from the Multiplexing Sample Preparation Oligonutide Kit of Illumina companies₀

Embodiment 1,40 aborted fetus tissues are carried out with chromosomal aneuploidies variation detect

1. DNA is extracted：

Above-mentioned 40 abortion tissue samples are extracted according to TiangenDP327-02Kit operating processes（Following cylinder claims, such as table 1) DNA, extracted DNA carries out building storehouse according to amended Illumina/Solexa standards Library development flow, the DNA of extraction is interrupted at random, choose the DNA fragmentation of 350bp-500bp magnitude ranges, and sequencing joint used is coupled with its two ends, each sample is coupled with different sequence labels, then hybridized with flowcell surface complementarities joint.One layer of single-stranded primer is connected with by flowcell surfaces, DNA fragmentation become it is single-stranded after by the primer base complementation with chip surface by one end " it is fixed, on chip；Other end（5, or 3) random and another Primers complementary nearby, also " and fixed, live, form " bridge（Bridge) ", 30 wheel amplification repeatedly, each unimolecule has obtained 1000 times of amplifications, as monoclonal DNA clusters.Then the sequence dna fragment that length is 150bp is obtained by double end sequencings on Illumina MiSeq.

Specifically, the about 100ng of above-mentioned abortion tissue sample DNA is will be obtained from, the Illumina/Solexa normal process after modifying builds storehouse, and idiographic flow is with reference to prior art（Referring to http：The Illumina/Solexa standards that 〃 www.mumina.com/ are provided build storehouse specification）.Determine that DNA library size and Insert Fragment are the sequencing that is available on the machine after about 250bp, QPCR accurate quantifications through 2100Bioanalyzer (Agilent).

2. sequencing：In the present embodiment, for being operated obtained from Cluster Station and MiSeq (PE sequencing) specification that above-mentioned 40 abortion tissue DNA samples are announced according to Illumina/Solexa officials, each sample is obtained about 100k data volumes and carry out upper machine sequencing, each sample is distinguished according to the sequence label.Using software SOAP2 is compared, by sequencing gained DNA sequence dna and the (hgl9 of version 37 in ncbi database;NCBI Build37) human genome reference sequences carry out it is not fault-tolerant compare, obtain positioning of the DNA sequence dna be sequenced on the genome.

3. data analysis

A) basic statistics：Unique aligned sequences number that every chromosome of statistics is fallen（The reads numbers uniquely compared）The percentage of genome sequence number is accounted for, Chr_N% is designated as, N represents chromosome numbers.

B) comparison set is calculated：The average Chr_N% of each bar chromosome of contrasting data collection is calculated, i.e., 0/rN_, following j represent check sample numbering, and N represents chromosome numbers.Wherein, for property

Chromosome is calculated with the standard control sample of identical sex respectively.The confidential interval of every chromosome 99% is calculated simultaneously.

C) Relative copy number is calculated：The relative copy rate of each bar chromosome of sample to be tested is calculated, D) threshold value and filtering：The copy rate that calculating is obtained is filtered.It is chromosome increase more than the confidential interval upper limit, is chromosome deficiency less than lower limit of confidence interval.

4. result is counted

For 40 samples, detailed testing result and the result is as shown in table 1 below.Wherein the result passes through comparative genome hybridization（Comparative Genomic Hybridization, CGH) chip draws.Reality-danger use Human Genome CGH Microarray Kit, (Agilent Technologies Inc.), the operation instruction fully according to manufacturer is operated.Fluorescence in situ hybridization technique is recycled to design corresponding probe (Fluorescence In Situ Hybridization, FISH), the FISHHER2 kits that this experiment is produced using Beijing Jin Pujia CGH chip results.

The aneuploidy testing result of 1. 40 samples of table

Sample number sequencing result CGH result FISH results

No. 2 three body 2, three bodies of repetition 2 of A350

No. 3 three body 3, three bodies of repetition 3 of A749

No. 3 three body 3, three bodies of repetition 3 of A221

No. 3 three body 3, three bodies of repetition 3 of A1599

No. 4 three body 4, three bodies of repetition 4 of A719

No. 4 three body 4, three bodies of repetition 4 of A230

No. 5 three body 5, three bodies of repetition 5 of A443

No. 5 three body 5, three bodies of repetition 5 of A1773

No. 6 three body 6, three bodies of repetition 6 of A1862

No. 6 three body 6, three bodies of repetition 6 of A1554

No. 7 three body 7, three bodies of repetition 7 of A520

No. 7 three body 7, three bodies of repetition 7 of A1757

No. 8 three body 8, three bodies of repetition 8 of A594

No. 8 three body 8, three bodies of repetition 8 of A1858

No. 9 three body 9, three bodies of repetition 9 of A1925

No. 10 L bodies of repetition of L bodies 10 of A1932 10

No. 10 L bodies of repetition of L bodies 10 of A385 10

No. 11 J disomes of repetition of J disomes 11 of A570 11

No. 12 L bodies of repetition of L bodies 12 of A382 12

No. 12 L bodies of repetition of L bodies 12 of A422 12

No. 13 L bodies of repetition of L bodies 13 of A352 13 No. 14 three body 14, three bodies of repetition 14 of A2064

No. 14 three body 14, three bodies of repetition 14 of A707

No. 14 three body 14, three bodies of repetition 14 of A236

No. 15 three body 15, three bodies of repetition 15 of A718

No. 16 three body 16, three bodies of repetition 16 of A1942

No. 16 three body 16, three bodies of repetition 16 of A233

No. 17 three body 17, three bodies of repetition 17 of A751

No. 17 three body 17, three bodies of repetition 17 of A240

No. 18 three body 18, three bodies of repetition 18 of A1838

No. 18 three body 18, three bodies of repetition 18 of A717

No. 20 three body 20, three bodies of repetition 20 of A1682

No. 21 three body 21, three bodies of repetition 21 of A225

No. 21 three body 21, three bodies of repetition 21 of A237

No. 22 three body 22, three bodies of repetition 22 of A254

No. 22 three body 22, three bodies of repetition 22 of A242

A2045 X Monosomies lack X monomers

A2043 X Monosomies lack X monomers

The monomers 21 of A1941 21 lack No. 21 monomers

The monomers 21 of A1764 21 lack No. 21 monomers

Thus, using the method for the present invention, the numerical abnormalities of chromosomes in biological specimen i.e. abortion tissue can effectively be determined.Industrial applicibility

The technical method of the present invention, can effectively determine the numerical abnormalities of chromosomes in biological specimen i.e. abortion tissue.Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change within protection scope of the present invention.The four corner of the present invention is provided by appended claims and its any equivalent.

In the description of this specification, the description of reference term " one embodiment ", " some embodiments ", " illustrative examples ", " example ", " specific example " or " some examples " etc. means to combine specific features, structure, material or the feature that the embodiment or example describe and is contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referring to the schematic representation of above-mentioned term.Moreover, specific features, structure, material or the feature of description can in an appropriate manner be combined in any one or more embodiments or example.

Claims

Claims

I, a kind of method for determining numerical abnormalities of chromosomes in biological specimen, it is characterised in that methods described includes：It is sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；

The sequencing data is compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；

Determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;

Determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;Based on formula ChrN%=N/M, the calculating ratio ChrN% of the predetermined chromosome is determined;And

Based on the calculating ratio ChrN% of the predetermined chromosome, determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.

2nd, according to the method described in claim 1, it is characterised in that the biological specimen is abortion tissue.

3rd, method according to claim 2, it is characterised in that the biological specimen is the fine hair of fetal tissue.

4th, according to the method described in claim 1, it is characterised in that further comprise from the biological specimen extract genomic DNA the step of,

Wherein, at least one extraction genomic DNA selected from salting out method, column chromatography and SDS methods is passed through.

5th, according to the method described in claim 1, it is characterised in that before the genomic DNA of the biological specimen is sequenced, the genomic DNA is interrupted at random.

6th, method according to claim 5, it is characterised in that random interrupt is by being carried out selected from least one of enzyme cutting method, atomization, ultrasonically treated and Hydroshear.

7th, method according to claim 5, it is characterised in that after the genomic DNA is interrupted at random, the length of DNA fragmentation is 200 ~ 300bp.

8th, according to the method described in claim 1, it is characterised in that the length of the sequencing data is 150bp.

9th, according to the method described in claim 1, it is characterised in that the reference gene group sequence is the human genome reference sequences of version 37 in ncbi database.

10th, according to the method described in claim 1, it is characterised in that use at least one of SOAP and BWA, the sequencing data is compared with the biological reference gene group sequence.

II, according to the method described in claim 1, it is characterised in that the calculating ratio ChrN% based on the predetermined chromosome, determines that the biological specimen further comprises for the predetermined chromosome with the presence or absence of numerical abnormalities of chromosomes：

The calculating ratio 01^% of the predetermined chromosome and mean_ChrN ° of characteristic rate/ratio of the predetermined chromosome are determined, to obtain the relative copy rate CR ChrN of predetermined chromosome described in the biological specimen, Wherein, if the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen exceeds the normal copy rate scope of the predetermined chromosome, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

12nd, method according to claim 11, it is characterized in that, the characteristic rate mean_ChrN% of the predetermined chromosome is determined by least 30 normal samples, and the normal copy rate scope of the predetermined chromosome is to be determined by calculating at least 30 normal samples for 99% confidential interval that the calculating ratio ChrN% of the predetermined chromosome is distributed.

13rd, according to the method described in claim 1, it is characterised in that the predetermined chromosome is selected from people No. 21, No. 13, No. 18, at least one of X and Y chromosome.

14th, according to the method described in claim 1, it is characterised in that the sequencing is by being carried out selected from least one of Roche/454 GS Junior, Illumina/MiSeq and Life Tecnologies/Ion Torrent PGM.

15th, a kind of computer-readable medium, it is characterised in that be stored with instruction on the computer-readable medium, the instruction is suitable to be executed by processor to determine numerical abnormalities of chromosomes in biological specimen through the following steps：

Obtain multiple sequencing datas of the genomic DNA of the biological specimen；

The sequencing data is compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；

Determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;

Determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;Based on formula ChrN%=N/M, the calculating ratio ChrN% of the predetermined chromosome is determined;And

Based on the calculating ratio ChrN% of the predetermined chromosome, determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.

16th, computer-readable medium according to claim 15, it is characterised in that the reference gene group sequence is the human genome reference sequences of version 37 in NCBI databases.

17th, computer-readable medium according to claim 15, it is characterised in that use at least one of SOAP and BWA, the sequencing data is compared with the biological reference gene group sequence.

18th, computer-readable medium according to claim 15, it is characterised in that the calculating ratio ChrN% based on the predetermined chromosome, determines that the biological specimen further comprises for the predetermined chromosome with the presence or absence of numerical abnormalities of chromosomes：

The calculating ratio 01^% of the predetermined chromosome and mean_ChrN ° of characteristic rate/ratio of the predetermined chromosome are determined, to obtain the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen,

Wherein, if the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen exceeds the normal copy rate scope of the predetermined chromosome, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

19th, computer-readable medium according to claim 18, it is characterised in that the aspect ratio of the predetermined chromosome Rate 1^＆^01^% is determined by least 30 normal samples.

20th, computer-readable medium according to claim 18, characterized in that, the normal copy rate scope of the predetermined chromosome is to be determined by calculating at least 30 normal samples for 99% confidential interval that the calculating ratio ChrN% of the predetermined chromosome is distributed.

21st, a kind of system for determining numerical abnormalities of chromosomes in biological specimen, it is characterised in that including：Sequencing device, the sequencing device is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；

Comparison device, the comparison device is connected with the sequencing device, for the sequencing data to be compared with the biological reference gene group sequence, to obtain the unique comparison sequencing data collection being made up of multiple unique comparison sequencing datas；Analytical equipment, the analytical equipment is connected with the comparison device, and whether there is numerical abnormalities of chromosomes for determining that the biological specimen is directed to predetermined chromosome,

Wherein, the analytical equipment further comprises：

First computing module, first computing module is used to determine unique number M for comparing unique comparison sequencing data included in sequencing data collection;

Second computing module, second computing module is used to determine unique number N for comparing sequencing data concentration from unique comparison sequencing data of predetermined chromosome;

3rd computing module, the 3rd computing module is used to be based on formula ChrN%=N/M, determines the calculating ratio ChrN% of the predetermined chromosome;And

Judge module, the judge module is used for the calculating ratio ChrN% based on the predetermined chromosome, determines that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome.

22nd, system according to claim 21, it is characterised in that the reference gene group sequence is the human genome reference sequences of version 37 in ncbi database.

23rd, system according to claim 21, it is characterised in that the comparison device uses at least one of SOAP and BWA, and the sequencing data is compared with the biological reference gene group sequence.

24th, systems approach according to claim 21, it is characterised in that the judge module is suitable to determine that the biological specimen whether there is numerical abnormalities of chromosomes for the predetermined chromosome by following：

The calculating ratio 01^% of the predetermined chromosome and mean_ChrN ° of characteristic rate/ratio of the predetermined chromosome are determined, to obtain the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen,

Wherein, if the relative copy rate CR_ChrN of predetermined chromosome described in the biological specimen exceeds the normal copy rate scope of the predetermined chromosome, it is determined that the biological specimen has numerical abnormalities of chromosomes for the predetermined chromosome.

25th, system according to claim 24, it is characterised in that the characteristic rate mean_ChrN% of the predetermined chromosome It is to be determined by least 30 normal samples, the normal copy rate scope of the predetermined chromosome is to be determined by calculating at least 30 normal samples for 99% confidential interval that the calculating ratio ChrN% of the predetermined chromosome is distributed.

26th, the system according to claim 21, it is characterised in that the sequencing device is selected from least one of Roche/454 GS Junior, Illumina/MiSeq and Life Tecnologies/Ion Torrent PGM.

27th, a kind of system for determining numerical abnormalities of chromosomes in biological specimen, it is characterised in that including：

Sequencing device, the sequencing device is used to be sequenced for the genomic DNA of the biological specimen, to obtain multiple sequencing datas；And

Computer-readable medium described in any one of claim 15 ~ 20.