CN105051208A - Method, system, and computer readable medium for determining base information of predetermined area in fetal genome - Google Patents

Method, system, and computer readable medium for determining base information of predetermined area in fetal genome Download PDF

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CN105051208A
CN105051208A CN201380074395.3A CN201380074395A CN105051208A CN 105051208 A CN105051208 A CN 105051208A CN 201380074395 A CN201380074395 A CN 201380074395A CN 105051208 A CN105051208 A CN 105051208A
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殷旭阳
蒋慧
陈盛培
龚淳
陈芳
张春雷
潘小瑜
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Chongqing Huada medical laboratory Co.,Ltd.
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BGI Shenzhen Co Ltd
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Abstract

A method, a system, and a computer readable medium for determining base information of a predetermined area in a fetal genome are provided. The method for determining base information of a predetermined area in a fetal genome comprises the following steps: obtaining a sequencing result of a DNA sample of a genome of an embryo cell, and a sequencing result of a genome sample of an individual related to embryo heredity; constructing a heredity sketch of the embryo based on the sequencing result of the DNA sample of the genome of the embryo cell, to determine an initial genotype of the embryo; determining haplotypes of parents of the embryo based on the sequencing result of the genome sample of the individual related to the embryo heredity; and using the initial genotype of the embryo as an observation sequence according to the hidden markov model, and determining the base information of the predetermined area in the fetal genome based on the haplotypes of the parents of the embryo.

Description

Method, system, and computer readable medium for determining base information of predetermined area in fetal genome
Determine the method for presumptive area base information, system and computer-readable medium priority information in embryonic gene group
Without technical field
The present invention relates to method, system and the computer-readable medium for determining presumptive area base information in embryonic gene group.Background technology
Genetic disease is due to the disease that inhereditary material changes and caused, with congenital, familial, lifelong participation and genetic feature.Genetic disease can be divided into 3 major classes：Single gene inheritance disease, disease of multifactorial inheritance and chromosome abnormality.The wherein many gene functions caused by the dominant or recessive inheritance of single Disease-causing gene of monogenic disease are abnormal；And disease of multifactorial inheritance is then the disease caused by multiple gene variations influence, it can be influenceed to a certain extent by outside environmental elements；Chromosome abnormality includes numerical abnormality and textural anomaly, and the most common is due to the Down's syndrome caused by No. 21 chromosome trisomy, and infant shows as other congenital features such as mongolism and limbs shape anomaly.Due to there is no effective therapeutic modality to genetic disease at present, treatment or medicine Slow solutions, somewhat expensive can only be pointedly supported, heavy economy and mental burden are brought to society and family.Therefore, just detect that ^ therefore good prevention work, to reach the purpose of prenatal and postnatal care, are very necessary to the disease state of child before birth in child.For example in vitro in reproductive process, before Embryonic limb bud cell uterus, the disease state of embryo is detected, will be significant.Genetic analysis are carried out to gamete or the embryo being implanted to before uterine cavity, the normal fetus implantation uterus of pathogenic factor will be excluded, so as to prevent the gestation of the child with hereditary disease.Mr. and Mrs with the pregnant genetic defect embryo of excessive risk are implemented with the detection of the disease state of preimplantation embryo, undoubtedly the birth of ill child can be effectively reduced, and many harm for avoiding selective abortion from being brought to pregnant woman and family.
However, current coherent detection means still have much room for improvement.The content of the invention
The present invention is the following discovery based on inventor and completed：
Inventor has found that it is limited the cell number detected can be sampled with biopsy in preimplantation embryo.For example cultivate to the blastomere of the 3rd day and only have 4-8 cell, only 1-2 cell of biopsy sampling can be carried out.Even in the Nang embryos of culture to the 5th day, 3-10 cell can only also be got by carrying out outer trophocyte's biopsy sampling.Because the amount of DNA of single or a small number of embryonic cells is limited, it is difficult to directly carry out comprehensive hereditary variation detection, it is necessary to pass through whole genome amplification（Whole Genome Amplification, WGA), enough amount of DNA are reached, comprehensive analysis could be done to hereditary variation.And whole genome amplification is commonly present Preference, the phenomenons such as certain heterozygous deletion or allele dropout (Allele Drop Out) can be produced, these all carry out risk for the related hereditary variation detection band of the hereditary disease of embryonic cell.
Therefore, the method that necessary exploitation can be positioned and corrected to the mistake such as allele dropout caused by amplification, to improve the accuracy of hereditary variation detection.
It is contemplated that at least solving one of technical problem present in prior art.Embryo's list is calculated by the inventive method Ploidy, can be positioned, and can be corrected to the site for occurring allele dropout.And embryo's haplotype analysis method can the hereditary information based on family member such as father and mother and propositus, or the hereditary information based on other embryonic cells from same father and mother calculated.
In one aspect of the invention, the present invention proposes a kind of method for determining presumptive area base information in embryonic gene group.It is that language infers with statistic algorithm to carry out the determination of embryo's haplotype that this method, which is combined, by the genotype of related individuals in family, that is, reviews the transmission of chromosome segment, haplotype state is inferred with reference to statistic algorithm.Embodiments in accordance with the present invention, this method comprises the following steps：Obtain embryonic cell genomic DNA sample sequencing result, and embryo genetic related individuals genome sample sequencing result；Based on the sequencing result of embryonic cell genomic DNA sample, the hereditary sketch of the embryo is built, to determine embryo's initial gene type；Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, determine the base information of presumptive area in embryonic gene group.
It should be noted that the genome forming process of filial generation, equivalent to the once random restructuring of parental gene group（I.e. it is chain exchange haplotype restructuring, and gamete random combine）.It regard the haplotype of embryo as hidden state（Hidden states), it regard the sequencing data after the unicellular whole genome amplification of embryo as observation sequence（), observations state transition probability is extrapolated by priori data using bayesian algorithm（Transition probabilities), build observation sequence probability distribution (observation symbol probabilities) and first:State is distributed (initial state distribution) without rate, ' and then algorithm compared by Hui Te（Viterbi algorithm) it can be inferred that most probable embryo's haplotype combination.Thus, embodiments in accordance with the present invention by hidden Markov model, for example, can compare algorithm by using Hui Te（Viterbi algorithm), with reference to the hereditary information of embryo genetic related individuals, it may be determined that the nucleotide sequence of specific region in embryonic gene group, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.For in embryonic cell whole genome amplification DNA sequencing result level of coverage it is relatively low, there is site or the DNA sequence dna of heterozygous deletion or allele dropout (Allele Drop Out), can exactly be inferred by this method and obtained.Therefore by this method, the site or sequence of heterozygous deletion or allele dropout (Allele Drop Out) can be corrected, makes testing result more accurately and reliably.
In still another aspect of the invention, the present invention proposes a kind of system for determining presumptive area base information in embryonic gene group.Just blunt according to embodiments of the invention, the system includes：Library construction device, the library construction device is suitable to the genomic DNA sample for embryonic gene group DNA sample and embryo genetic related individuals, and sequencing library is built respectively；Sequencing device, the sequencing device is connected with the library construction device, and suitable for the sequencing library is sequenced, to obtain the sequencing result of embryo and embryo genetic related individuals；And analytical equipment, the analytical equipment is connected with the sequencing device, and is suitable to：Based on the sequencing result of embryonic gene group DNA sample, the hereditary sketch of the embryo is built, to determine embryo's initial gene type；Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, determine the base information of presumptive area in embryonic gene group.Embodiments in accordance with the present invention, can further include embryo biopsy device, and the embryo biopsy device is suitable to carry out biopsy for the embryo of in vitro culture, embryonic cell is sampled.Using the system, it can effectively implement the method for presumptive area base information in foregoing determination embryonic gene group, for example can compare algorithm by using Hui Te by hidden Markov model（Viterbi algorithm), with reference to the hereditary information of embryo genetic related individuals, it may be determined that the nucleic acid sequence of specific region in embryonic gene group Row, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection, are determined so as to the hereditary information effectively to embryonic gene group.
In another aspect of this invention, the invention also provides a kind of computer-readable medium.Embodiments in accordance with the present invention, based on the sequencing result of embryonic cell genomic DNA sample, build the hereditary sketch of embryo, to determine embryo's initial gene type；Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And so that according to hidden Markov model, embryo's initial gene type, based on the haplotype of the embryo father and mother, determines the base information of presumptive area in embryonic gene group as observation sequence.Utilize the computer-readable medium of the present invention, the instruction of its storage can be effectively executed by processor, so as to by hidden Markov model, for example can be by using Hui Te than algorithm (Viterbi algorithm), sequencing result based on embryonic cell, with reference to the hereditary information of embryo genetic related individuals, it may be determined that the nucleotide sequence of specific region in embryonic gene group, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
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 is the schematic flow sheet analyzed using hidden Markov model according to one embodiment of the invention；And Fig. 2 is the structural representation for being used to determine the system of presumptive area nucleotide sequence in embryonic gene group according to one embodiment of the present of invention.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 ", " second " are 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 ".Further, in the description of the invention, unless otherwise indicated, " multiple " are meant that two or more.
The method for determining presumptive area information in embryonic gene group
In the first aspect of the present invention, the present invention proposes a kind of method for determining presumptive area base information in embryonic gene group.Embodiments in accordance with the present invention, this method comprises the following steps：
First, obtain embryonic cell genomic DNA sample sequencing result, and embryo genetic related individuals genome sample sequencing result.Wherein, term " embryo genetic related individuals " used in herein is referred in hereditary meaning, has akin individual between embryo, such as embodiments in accordance with the present invention, " the embryo genetic related individuals " that can be used for parental generation such as father and mother, grand parents, grand parents and the embryo father and mother of embryo other children.It is mentioned here Other children of embryo father and mother should be interpreted broadly, and both can be the children or still unborn children (embryonated egg or embryo being born）Or dead embryo or embryo or the embryonated egg of in vitro culture, as long as having identical father and mother with embryo to be detected.
Embodiments in accordance with the present invention, the source of embryonic cell genomic DNA sample is not particularly restricted.Embodiments in accordance with the present invention, specifically, can carry out biopsy sampling for embryo, obtain embryonic cell, and carry out whole genome amplification (WGA) to embryonic cell sample, to obtain embryonic cell genomic DNA.The term " embryo biopsy " used herein refers to utilize aobvious ^:Technology, the separate section embryonic cell from embryo, or from embryonated egg/gamete separate section cell technology.Wherein, embodiments in accordance with the present invention, the embryonic cell may be from any of blastomere, Nang embryos trophoderm, embryonated egg and gamete, can be individual cells or 2-10 cell.In addition it is also possible to whole genome amplification be carried out using any pregnant woman's sample containing embryo's nucleic acid, to obtain embryonic gene group DNA sample.Thus, it is possible on the premise of embryo's normal development is not influenceed, effectively be monitored to the genome of embryo.Wherein, " whole genome amplification (WGA) " mainly includes multiple strand displacement amplification (Multiple Displacement Amplification, MDA) and PCR-based WGA technologies, flow can be expanded using the WGA of independent development, can also be using the kit serial REPLI-g of commercialized kit such as Qiagen companies, the GenomePlex WGA kits (WGA4) of Sigma- Aldriches, the PicoPlex WGA kits of Rubicon Genomics companies, illustra Genomiphi WGA kits of GE-Healthcare companies etc..After DNA amplification sample, the genomic DNA sample of DNA amplification sample and embryo genetic related individuals for embryonic cell builds sequencing library respectively.
On for sample of nucleic acid, build the method and flow of sequencing library, those skilled in the art can suitably be selected according to different sequencing technologies, details on flow, it may refer to be sequenced manufacturer's code that for example Illumina companies are provided of instrument, for example, see Illumina companies Multiplexing Sample Preparation Guide (Part#l 005361;Feb 2010) or Paired-End Sample Prep Guide (Part#l 005063；Feb 2010), by referring to be incorporated into herein.Embodiments in accordance with the present invention, the method and apparatus that sample of nucleic acid is extracted from biological specimen, are also not particularly limited, and can be carried out using the nucleic acid extraction kit of commercialization.
After sequencing library is built, sequencing library is applied to sequencing instrument, sequencing library is sequenced, and obtain corresponding sequencing result, the sequencing result is made up of multiple sequencing datas.Embodiments in accordance with the present invention, the method and apparatus that can be used for being sequenced is not particularly restricted, including but not limited to dideoxy chain termination；It is preferred that high-throughout sequence measurement, the characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, sequencing efficiency is further improved.Subsequently sequencing data is analyzed thus, it is possible to improve, especially statistical check analysis when accuracy and the degree of accuracy.The high-throughout sequence measurement includes but is not limited to second generation sequencing technologies either single-molecule sequencing technology.Second generation microarray dataset (the Jan of Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010;ll(l):31-46) include but is not limited to Illumina- Solexa (GA, HiSeq2000, Miseq etc.；), ABI-SOLiD, Life Technologies-Ion Torrent/Proton and Roche-454 (burnt upright stone tablet acid sequencing）Microarray dataset；Single-molecule sequencing platform (technology）The including but not limited to true single-molecule sequencing technology of Helicos companies（True Single Molecule DNA sequencing), Pacific Biosciences companies unimolecule is sequenced (single molecule real-time (SMRT)) in real time, and Oxford Nanopore Technologies companies nano-pore sequencing technology etc.（Rusk, Nicole (2009-04-01). Cheap Third-Generation Sequencing. Nature Methods 6 (4): 244-245 ).With sequencing The continuous evolution of technology, skilled artisans appreciate that be that genome sequencing can also be carried out using other sequence measurements and device.It is just blunt according to specific example of the invention, it is possible to use the genome sequencing library is sequenced at least one selected from Illumina-Solexa, ABI-SOLiD, Life Technologies-Ion Torrent/Proton, Roche-454 and single-molecule sequencing device.
Embodiments in accordance with the present invention, can the sequencing result based on embryonic cell genomic DNA sample, the hereditary sketch of the structure embryo, to determine embryo's initial gene type after sequencing result is obtained.Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined.Embodiments in accordance with the present invention, by the way that the sequencing result of embryonic cell genomic DNA sample is compared with reference sequences, build the hereditary sketch of the embryo.By the way that the sequencing result of the embryo genetic related individuals genome sample is compared with reference sequences, the genotype of the embryo genetic related individuals is determined；And the genotype based on the embryo genetic related individuals, determine the haplotype of the embryo father and mother.Embodiments in accordance with the present invention, can be used as reference sequences using known mankind's reference gene group.For example, embodiments in accordance with the present invention, the mankind's reference gene group used is NCBI 36.3, HG18.In addition, embodiments in accordance with the present invention, the method being compared is not particularly restricted.According to a particular embodiment of the invention, it can be compared using SOAP.
Finally, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, according to hidden Markov model, the base information of presumptive area in embryonic gene group is determined.
Term " presumptive area " used in herein should broadly understood, and refer to any comprising the region that may occur the nucleic acid molecules in scheduled event site.For snp analysis, the region for including SNP site can be referred to.For analyzing chromosomal aneuploidy, then presumptive area refers to total length or the part of the chromosome to be analyzed, that is, selects all sequencing datas from the chromosome.The method of the sequencing data from respective regions is selected from sequencing result to be not particularly limited.Embodiments in accordance with the present invention, can be by the way that resulting all sequencing datas be compared with known nucleic acid reference sequences, so as to obtain coming from the sequencing data of presumptive area.In addition, embodiments in accordance with the present invention, presumptive area can also be discontinuous multiple spaced points on genome.Embodiments in accordance with the present invention, the type for the reference sequences that can be used is not particularly restricted, and can be any known array containing area-of-interest.
Embodiments in accordance with the present invention, with reference to the hereditary information of embryo genetic related individuals, according to hidden Markov model, can determine the base information of the presumptive area by the sequencing result based on the embryonic cell.Embodiments in accordance with the present invention, can compare algorithm by hidden Markov model using Hui Te（Viterbi algorithm), determine the base information of specific region in embryonic gene group.Thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
Below with reference to Fig. 1, to being described in detail by hidden Markov model using Hui Te than the principle that algorithm is analyzed：As previously described, term " embryo genetic related individuals " used in herein is referred in hereditary meaning, has akin individual between embryo, such as embodiments in accordance with the present invention, " the embryo genetic related individuals " that can be used for parental generation such as father and mother, grand parents, grand parents and the embryo father and mother of embryo other children.Other children of embryo father and mother mentioned here should be interpreted broadly, and both can be the children or still unborn children being born（Embryo or embryonated egg）Or dead embryo or embryo or the embryonated egg of in vitro culture, as long as having identical father and mother with embryo to be detected.
Thus, the genome forming process of filial generation, equivalent to the once random restructuring of parental gene group（I.e. it is chain exchange haplotype restructuring, and gamete random combine）.It regard the haplotype of embryo as hidden state（Hidden states), by embryo Sequencing data after unicellular whole genome amplification is used as observation sequence（), observations state transition probability is extrapolated by priori data（Transition probabilities), build and determine observation sequence probability distribution (observation symbol probabilities) and initial state probabilities distribution（Initial state distribution), algorithm is then compared by Hui Te（Viterbi algorithm) it can be inferred that most probable embryo's haplotype combination.Thus, embodiments in accordance with the present invention by hidden Markov model, for example, can compare algorithm by using Hui Te（Viterbi algorithm), with reference to the hereditary information of embryo genetic related individuals, it may be determined that the nucleotide sequence of specific region in embryonic gene group, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
Labor step is as follows：
Mark：
I. the number of sites for needing detection is N.
II. observation sequence；
III. hidden state collection is：" -1 ", father and mother any bar chromosomal inheritance is defined to filial generation.0 represents that chromosome for having entailed the positive persons of first i, and 1 represents that chromosome for not entailing propositus.
IV. observer state collection is：' _ 1 ", ^, 1 represent entail propositus chromosome it is consistent with embryo genetic sketch genotype, 0 represent it is inconsistent.The first step, builds initial state probabilities distribution vector, monoploid restructuring transfer matrix and observation sequence probability matrix
I. initial state probabilities distribution is designated as ^^^) ^, ' ^0'5),
In the case of without reference to data, it can be assumed that the possibility that every kind of hidden state occurs is equal.
II. note monoploid restructuring transfer matrix is designated asA ={ }, (z,e, i.e., the probability that hidden state is shifted. l-Nr I Np i = j
Nr, Np are represented to expect restructuring number and mononucleotide polymorphism site number respectively, be can use the natural number between 20-40 according to priori data Nr, for calculating the transition probability of hidden state, that is, calculate the probability that the haplotype of each base composition is recombinated.
III. note observation sequence probability matrix isβ =(^, G ≡ S, k eV)5Here probability distribution filial generation must be homozygosis（) or must to be the site of heterozygosis (Must-het) estimate Must-hom.
#sites(L > 0, Must-hom.)
¥0)
#w'to(L>0, Must-het. or Must-hom.) b!(l)=1-(0) second step, builds local probability matrix and the adopted office's Shao's probability of reverse pointer Shu： te{l...N) i(j) = argmaxSt_1(i)-a
Define reverse pointer：{ l-N } the 3rd step, determines end-state state, and recall optimal path
qN* =argmaxSN(qN)
Determine end-state,.Recall optimal path, i.e. most probable embryo single-gene typeqt4th step, i.e., embodiments in accordance with the present invention, according to hidden Markov model, determine that the base information of presumptive area in embryonic gene group further comprises to output result：
Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
According to specific embodiment, the hidden Markov model uses following parameters：Initial state probabilities are distributed as=and ^,£5'^ = 0-5)
Hidden state transfer probability matrix beλ = eSWherein,
Nr, Np represent to expect respectively restructuring number and mononucleotide polymorphism site number, and Nr is natural number, span 20-40, and observation sequence probability matrix iss ={) }, G'eS, eV), wherein,
#sites(L > 0,Must-hom.)
# sites ( L >0, Must-het. or Must-horn.) b. (i)=l-b. (o)
#sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation；Office's Shao's probability is ieil-Wj, and reverse pointer is te{l...N} ^
qN* =argmaxSN(qN)
Yi Huite is through recursive call end-state than algorithm
Recall optimal path, determine the base letter of most probable embryo's presumptive area, cease and be=Ψ"ι(^ + 1) (=1,2,3 ..., N_1) Wherein, term " local probability () " used herein above and " reverse pointer Ψ;() " is all the classical definition for continuing to use Viterbi algorithms.On the detailed description of the definition of the parameter, may refer to Lawrence R. Rabiner, PROCEEDINGS OF THE 2 months IEEE, Vol.77, No.2,1989 years, by referring to be incorporated by herein.
Analyzed thereby, it is possible to the sequence effectively to embryonic gene group.Compared to detection technique method before other existing implantation, this method has following technical advantage, is mainly reflected in accuracy and obtainable hereditary information amount：
1) according to embodiments of the present invention, for the father source property site of embryo and Disease in Infants site, it can detect well, Detection accuracy may be up to more than 95%, and can detect a variety of variation types, expand the scope of disease detection.
2) according to embodiments of the present invention, for some embryo is unicellular or few cells whole genome amplification DNA sequencing result in level of coverage it is relatively low, there is site or the DNA sequence dna of heterozygous deletion or allele dropout (Allele Drop Out), can exactly be inferred by this method and obtained.Therefore by this method, the site or sequence of heterozygous deletion or allele dropout (Allele Drop Out) can be corrected, makes testing result more accurately and reliably.
3) genetic disease mapping according to embodiments of the present invention, can be carried out, for some chain relevant diseases, can be directly inferred to by the information in other sites, it is once obtainable to contain much information, more there is directive significance to clinical detection.
In addition, according to embodiments of the present invention, the method of presumptive area base information in the determination embryonic gene group of the present invention, it is not limited only to a certain genetic polymorphism site such as SNP or STR, it is applicable to all genetic polymorphism sites, and can simultaneously be used with a variety of sites, so as to checking mutually.System for determining presumptive area information in embryonic gene group
In still another aspect of the invention, the present invention proposes a kind of system for determining presumptive area nucleotide sequence in embryonic gene group.Embodiments in accordance with the present invention, with reference to Fig. 2, the system 1000 can include：Library construction device 100, sequencing device 200 and analytical equipment 400.
Embodiments in accordance with the present invention, library construction device 100 is suitable to the genomic DNA sample for embryonic gene group DNA sample and embryo genetic related individuals, and sequencing library is built respectively.Embodiments in accordance with the present invention, sequencing device 200 is connected with library construction device 100, and suitable for the sequencing library is sequenced, to obtain the sequencing result of embryo and embryo genetic related individuals.It is just blunt according to embodiments of the invention, it can further include DNA sample separator and DNA amplification devices（Not shown in figure）, the DNA sample separator for embryo suitable for carrying out biopsy sampling, acquisition embryonic cell.Embryonic cell may be from any of blastomere, Nang embryos trophoderm, embryonated egg and gamete, can be individual cells or ^ Jiao amounts such as 2-10 cell of cell, it would however also be possible to employ any pregnant woman's sample containing embryo's nucleic acid.The DNA amplification devices are suitable to sample the embryonic cell obtained progress whole genome amplification for biopsy, are analyzed with obtaining sufficient amount DNA for subsequent detection.Thus, it is possible on the premise of embryo's normal development is not influenceed, effectively be monitored to the genome of embryo.On the method and flow of whole genome amplification, mainly including multiple strand displacement amplification（Multiple Displacement Amplification, MDA) and PCR-based WGA technologies, can using independent development WGA amplification flows, it would however also be possible to employ serial the REPLI-g of commercialized kit such as Qiagen companies kit, the GenomePlex WGA kits (WGA4) of Sigma- Aldriches, the PicoPlex WGA kits of Rubicon Genomics companies, illustra Genomiphi WGA kits of GE-Healthcare companies etc.. On for sample of nucleic acid, build the method and flow of sequencing library, those skilled in the art can suitably be selected according to different sequencing technologies, details on flow, it may refer to be sequenced manufacturer's code that for example Illumina companies are provided of instrument, for example, see Illumina companies Multiplexing Sample Preparation Guide (Part#l 005361;Feb 2010) or Paired-End SamplePrep Guide (Part#l 005063；Feb 2010), by referring to be incorporated into herein.Embodiments in accordance with the present invention, the method and apparatus that sample of nucleic acid is extracted from biological specimen, are also not particularly limited, and can be carried out using the nucleic acid extraction kit of commercialization.Embodiments in accordance with the present invention, the method and apparatus that can be used for being sequenced is not particularly restricted, including but not limited to dideoxy chain termination；It is preferred that high-throughout sequence measurement, the characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, sequencing efficiency is further improved.Subsequently sequencing data is analyzed thus, it is possible to improve, especially statistical check analysis when accuracy and the degree of accuracy.The high-throughout sequence measurement includes but is not limited to second generation sequencing technologies either single-molecule sequencing technology.The second generation microarray dataset（ Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010 Jan;ll(l):31-46) include but is not limited to Illumina-Solexa (GA, HiSeq, Miseq etc.）, ABI-SOLiD, Life Technologies-Ion Torrent/Proton and Roche-454 (pyrosequencings）Microarray dataset；Single-molecule sequencing platform（Technology）The including but not limited to true single-molecule sequencing technology (True Single Molecule DNA sequencing) of Helicos companies, Pacific Biosciences companies unimolecule is sequenced (single molecule real-time (SMRT)) in real time, and Oxford Nanopore Technologies companies nano-pore sequencing technology etc.（ Rusk, Nicole (2009-04-01). Cheap Third-Generation Sequencing. Nature Methods 6 (4): 244-245 ).With the continuous evolution of sequencing technologies, skilled artisans appreciate that be that genome sequencing can also be carried out using other sequence measurements and device.According to the specific example of the present invention, it is possible to use the genome sequencing library is sequenced at least one selected from Illumina-Solexa, ABI-SOLiD, Life Technologies-Ion Torrent/Proton, Roche-454 and single-molecule sequencing device.
Embodiments in accordance with the present invention, can also include comparison device 300.Embodiments in accordance with the present invention, comparison device 300 is connected with sequencing device 200, and suitable for resulting sequencing result and reference sequences are compared, the sequencing result of embryonic cell genomic DNA sample is compared with reference sequences with will pass through, the hereditary sketch of the embryo is built；By the way that the sequencing result of the embryo genetic related individuals genome sample is compared with reference sequences, the genotype of the embryo genetic related individuals is determined；And the genotype based on the embryo genetic related individuals, determine the haplotype of the embryo father and mother.Embodiments in accordance with the present invention, the type for the reference sequences that can be used is not particularly restricted, and can be any known array containing area-of-interest.Embodiments in accordance with the present invention, can be used as reference sequences using known mankind's reference gene group.For example, embodiments in accordance with the present invention, the mankind's reference gene group used is NCBI 36.3, HG18.Another sunset is foretold, and embodiments in accordance with the present invention, the method being compared is not particularly restricted.According to a particular embodiment of the invention, it can be compared using SOAP.
Embodiments in accordance with the present invention, analytical equipment 400 is suitable to：Based on the sequencing result of embryonic cell genomic DNA sample, the hereditary sketch of the embryo is built, to determine embryo's initial gene type；Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, determine the base information of presumptive area in embryonic gene group.
Embodiments in accordance with the present invention, according to hidden Markov model, determine the base information of presumptive area in embryonic gene group Further comprise：
Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
According to specific embodiment, the hidden Markov model uses following parameters：
Initial state probabilities are distributed as=£5'^=0-5),
Nr, Np represent expectation restructuring number and mononucleotide polymorphism site number respectively, and Nr can use the natural number between 20-40, and observation sequence probability matrix iss =^) } G'eS eV), wherein
# sites(L > 0,Must-hom.)
b,(0)
# sites ( L > 0 Must-het. or Must-horn.) ; ( 1) = 1— b; ( 0)
#sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation；
P # ^ ^ ( J ) = max - 1 ( ) · aij] -bj(K) / F fl Νλ
Office's Shao's probability is ieil-Wj,
Xi;(j) = argmaXi5'i-i( -¾ ti
Reverse pointer for '
qN* =argmaxSN(qN)
Recursive call end-state is to recall optimal path, and the base information for determining most probable embryo's presumptive area is
(i=123... N_1 term local probabilities used herein above and reverse pointer Ψ;() is all the classical definition for continuing to use Viterbi algorithm.On the detailed description of the definition of the parameter, may refer to Lawrence R. Rabiner, PROCEEDINGS OF THE 2 months IEEE, Vol.77, No.2,1989 years, by referring to be incorporated by herein.
On data analysis component, before have been carried out be described in detail, also of course be applied to determination embryonic gene group presumptive area nucleotide sequence system.Repeat no more.
Thus, using the system, it can effectively implement the method for presumptive area nucleotide sequence in foregoing determination embryonic gene group, algorithm can be compared for example, by Hui Te by hidden Markov model（Viterbi algorithm) decoding, the base information of specific region in embryonic gene group is determined, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
In addition, embodiments in accordance with the present invention, presumptive area is the known site that there is genetic polymorphism, and genetic polymorphism is at least one selected from SNP and STR. Described term " connected " should broadly understood herein, both can be to be joined directly together or be indirectly connected to, as long as the linking in above-mentioned functions can be realized.
It should be noted that, skilled artisans appreciate that, the system that the feature and advantage of the method for presumptive area nucleotide sequence are also suitable for determining presumptive area nucleotide sequence in embryonic gene group in determination embryonic gene group described above, for convenience of description, is no longer described in detail.Computer-readable medium
In still another aspect of the 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, it is described instruction be suitable to be executed by processor so as to：
Based on the sequencing result of embryonic cell genomic DNA sample, the hereditary sketch of embryo is built, to determine embryo's initial gene type；
Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, the haplotype based on the embryo father and mother determines the base information of presumptive area in embryonic gene group.
Thus, using the computer-readable medium, foregoing method can effectively be implemented, so as to compare algorithm for example, by Hui Te（Viterbi algorithm), by hidden Markov model, the base information of specific region in embryonic gene group is determined, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
Embodiments in accordance with the present invention, according to hidden Markov model, determine that the base information of presumptive area in embryonic gene group further comprises：
Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
According to specific embodiment, the hidden Markov model uses following parameters：
Initial state probabilities are distributed as=£ 5'^ = 0-5) , l - Nr l Np i = j
A = { aij, the probability matrix of (i, j e S) ^ wherein Nr/Np i ≠ j hidden states transfer is
Nr, Np represent expectation restructuring number and mononucleotide polymorphism site number respectively, and Nr can use the natural number between 20-40, and observation sequence probability matrix is its towel
# sites (L > 0,Must-hom.)
b, (o)
# sites ( L >0, Must-het. or Must-horn.); ( 1) = 1— b; ( 0)
#sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation； Office's Shao's probability is ieil-Wj
Ψ, (_/·) = arg max a
Reverse pointer is te { l...N } ^
qN* =argmaxSN(qN)
It is backtracking optimal path through recursive call end-state, determines most probable embryo fate, the base information in domain is=Ψ"ι(^ + 1) (=1,2,3 ..., N_1) term local probability () used herein above and reverse pointer Ψ;() is all the classical definition for continuing to use Viterbi algorithm.On the detailed description of the definition of the parameter, may refer to Lawrence R. Rabiner, PROCEEDINGS OF THE 2 months IEEE, Vol.77, No.2,1989 years, by referring to be incorporated by herein.
On data analysis component, before have been carried out be described in detail, also of course be applied to determination embryonic gene group presumptive area nucleotide sequence system.Repeat no more.
Thus, using the system, it can effectively implement the method for presumptive area nucleotide sequence in foregoing determination embryonic gene group, algorithm can be compared for example, by Hui Te（Viterbi algorithm), by hidden Markov model, the base information of specific region in embryonic gene group is determined, thus, it is possible to which effectively the hereditary information to embryonic gene group carries out being implanted into preceding detection.
In addition, embodiments in accordance with the present invention, presumptive area is the known site that there is genetic polymorphism, and genetic polymorphism is at least one selected from SNP and STR.
For the purpose of this specification, " computer-readable medium " can any can be included, store, communicating, propagating or transmission procedure for instruction execution system, device or equipment or combines these instruction execution systems, device or equipment and the device used.The more specifically example of computer-readable medium（Non-exhaustive list）Including following：Electrical connection section with one or more wirings（Electronic installation）, portable computer diskette box（Magnetic device）, random access memory（), RAM read-only storage (ROM), erasable edit read-only storage（EPROM or flash memory）, fiber device, and portable optic disk read-only storage（CDROM).In addition, computer-readable medium, which can even is that, to print the paper or other suitable media of described program thereon, because for example can be by carrying out optical scanner to paper or other media, then enter edlin, interpret or handled electronically to obtain described program with other suitable methods if necessary, be then stored in computer storage.
It should be appreciated that each several part of the present invention can be realized with hardware, software, firmware or combinations thereof.In the above-described embodiment, multiple steps or method can be performed in memory and by suitable instruction execution system with storage software or firmware is realized.If for example, being realized with hardware, with another embodiment, can be realized with any one of following technology well known in the art or their combination：Discrete logic with the logic gates for realizing logic function to data-signal, the application specific integrated circuit with suitable combinational logic gate circuit, programmable gate array（), PGA field programmable gate array（FPGA) etc..
Those skilled in the art are appreciated that all or part of step for realizing above-described embodiment method carrying can be by program to instruct the hardware of correlation to complete, and described program can be stored in a kind of computer-readable recording medium, The program upon execution, including one or a combination set of the step of embodiment of the method.
In addition, each functional unit in each of the invention embodiment can be integrated in a processing module or unit is individually physically present, can also two or more units be integrated in a module.Above-mentioned integrated module can both be realized in the form of hardware, it would however also be possible to employ the form of software function module is realized.If the integrated module is realized using in the form of software function module and as independent production marketing or in use, can also be stored in a computer read/write memory medium.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.Agents useful for same or the unreceipted production firm person of instrument, are that be able to can for example be purchased from Illumina companies by the conventional products of acquisition purchased in market.
Conventional method：
With reference to Fig. 1, the key step of the embodiment of the present invention includes：
1) unicellular or few cells sample, the 3rd day spilting of an egg glomus cell or the sampling of Nang embryo trophocytes biopsy in the 5th day, or embryonated egg sampling, or other unicellular or few cells samplings are obtained.
2) unicellular or few cells sample carries out whole genome amplification, and amplified production DNA builds library according to new-generation sequencing Platform Requirements, and is sequenced.
3) unicellular or few cells sequencing data is compared after filtering with human genome reference sequences.
4) Preliminary detection of the hereditary variation such as embryonic cell SNP and missing repetition is carried out according to comparison result.
5) the hereditary sketch of embryonic gene group is built, embryonic gene type is initialized.
6) embryo genetic related individuals are collected, such as family member such as father and mother and propositus, and/or grand parents, grand parents etc. or with other embryo's samples of a pair of father and mother, extraction genomic DNA, library is built according to new-generation sequencing Platform Requirements, and is sequenced.
7) embryo genetic related individuals DNA sequencing data are filtered, and are compared with human genome reference sequences.
8) according to comparison result, analysis determines the genotype of the genetic correlations such as propositus, father and mother individual.
9) using the genotype of the genetic correlations such as propositus, father and mother individual, the haplotype of parents is inferred.
10) by HMM decoding process, embryo's haplotype is inferred with father and mother's haplotype result.
11) the final determination of embryo genetic variation.
With reference to Fig. 1, the HMM and labor step that information analysis part is used in the embodiment of the present invention are as follows：
Mark：
1st, it is N to need the number of sites detected.
2nd, observation sequence：
3rd, hidden state collection is：Two ^^,：^, defines father and mother which bar chromosomal inheritance to filial generation.0 representative has entailed elder generation That chromosome of card person.
4th, observer state collection is：V={ 0,1 }.The chromosome that 1 representative entails propositus is consistent with embryo genetic sketch genotype.
0 represent it is inconsistent.The first step, builds initial state probabilities distribution vector, monoploid restructuring transfer matrix and observation sequence probability matrix
I. initial state probabilities distribution is designated as； r = {； r;, (i ≡ S, ^=0.5) in the case of without reference to data, if the possibility that every kind of hidden state occurs is equal.
II. note monoploid restructuring transfer matrix be designated as A=, (z'eS), i.e. hidden state transfer probability.
Nr, Np represent to expect restructuring number and mononucleotide polymorphism site number respectively.
Build observation sequence probability matrix
It is S to remember observation sequence probability matrix Here probability distribution filial generation must be homozygosis
Or must to be the site of heterozygosis (Must-het) estimate (Must-horn).
# sites L >0, Must-het. or Must-horn. second steps, build local probability matrix, and reverse pointer
Define local probability：St (j)=max [St aij]-bj (K) te { l...N } define reverse pointer： Ψ, ( j) = arg max 5t_x(i) steps of-a.. t { l...N } the 3rd, determine end-state state, and recall optimal path
Determine end-state, qN* = arg max SN(qN) backtracking optimal path, i.e. most probable embryo single-gene type *=ψί+1(^ί + 1) (=l, 2,3 ..., N- 1)
4th step, output result embodiment 1
First, sample collection and processing：
A couple once gave birth to the daughter Α (propositus) of a severe beta Thalassemia, therefore received IVF (joint embryo transfer technologies in vitro fertilization）- PGD is treated, it would be desirable to give birth to a healthy baby being consistent with propositus's HLA distribution type again.By an IVF-PGD treatment cycle, the unicellular biopsy sampling of blastomere embryos was carried out at the 3rd day, the blastomere after biopsy continued to cultivate to the 5th day Nang embryonic stage, carried out Nang embryo vitrificated cryopreserrations.The unicellular sample of blastomere that biopsy is obtained is carried out The whole genome amplification (- OWGA of PED7- 1), whole genome amplification uses the REPLI-g MDA amplification kits of Qiagen companies, is strictly operated according to kit specification, and amplified production is used for new-generation sequencing and other analyses.The peripheral blood of father (PED4-F), mother (PED3-M) and daughter propositus (PED-Sister) in this family are also collected simultaneously, extract genomic DNA.
After IVF-PGD treatments are completed, select after correct Embryonic limb bud cell uterus, successful pregnancy, amniotic fluid of pregnant woman (PED7-2-OAF) can be extracted after 14 weeks, or collects embryo's Cord blood, DNA is extracted, it is sequenced, to verify the genotyping result of embryonic cell.The unicellular whole genome amplification product DNA of embryo interrupts instrument with Covaris and interrupted to the fragment of 200bp sizes, according to the upper confidential library warp asked progress to build storehouse, built of illumia companies HiSeq2000 sequenators
Agilent Bioanalyzer 2100 and Q-PCR methods carry out Quality Control, and qualified library proceeds illumina
HiSeq2000 sequencers, sequencing strategy is the index of Pair End 90 (i.e. two-way 90bp index sequencings；), depth 30X, genome sequencing is sequenced.The parameter setting and operating method of its Instrumental are all according to illumina operation manuals（Can be by http://wwwillumina.com/support/documentation.ilmn is obtained).Father, mother and daughter's propositus peripheral blood DNA, and amniotic fluid of pregnant woman or embryo's Cord blood DNA interrupt instrument with Covaris and interrupted to the fragment of 200bp sizes, upper according to illumina companies HiSeq2000 sequenators confidential asks progress to build storehouse, library carries out the capture of All in One chips, and concrete operations are shown in that the capture of NimbleGen SeqCap EZ chips builds storehouse and illustrates (http://www.nimblegen.com/products/seqcap/ez/index.html can be obtained).All in One chips are the target area capture chips of autonomous Design, and its target area includes full exon region, 1M SNP site region and mhc gene area.Library after chip capture carries out Quality Control through Agilent Bioanalyzer 2100 and Q-PCR methods, qualified library proceeds illumina HiSeq2000 sequencers, sequencing strategy is the index of Pair End 90, and sequencing depth is the 30-50X of target area.The parameter setting and operating method of its Instrumental are all according to illumina operation manuals（Can be by http://wwwillumina.com/support/documentation.ilmn is obtained).2nd, sequencing data is analyzed：
1st, propositus and parent gene group DNA sequencing parting：
A) sequence of low quality value and joint pollution is filtered out
B) Burrows-Wheeler Aligner (BWA, http/bio-bwa.sourceforge.net/) ^ is used)!It is " high-quality：Ill sequences sequence alignment is in mankind's reference gene group（Hgl9, NCBI release GRCh37).And the sequence of PCR repeat amplification protcols is got rid of with SAMtools.
C) using Genome Analysis Toolkit (GATK, http：〃 www.broadinstitute.org/gatk/) calibrate base mass value and carry out the detection that single base nucleotide polymorphisms and missing are repeated.
2nd, the hereditary sketch of embryonic gene group is built.
3rd, the haplotype of father and mother is inferred according to propositus's genotype：
Infer haplotype according to core families, specifically refer to document B. L. Browning and S. R. Browning. Am J Hum Genet 84:210-223. (2009) carry out. 4th, the haplotype of embryonic cell is inferred according to father and mother's haplotype：
A) the unicellular sequencing data of embryo is compared using BWA and arrives mankind's reference gene group（Hgl9, NCBI release GRCh37)
B) initial state probability vector, and embryo monoploid restructuring transfer matrix are built, Nr=30 are other by upper described.C) sequencing information in each site of statistics embryonic cell, and build observation sequence probability matrix（By upper described）.
D) local probability matrix, and reverse pointer are built（By upper described）.
E) end-state is determined, and recalls optimal path
F) export
G) using the postnatal amniotic fluid genotypic results of embryo as reference, the Genotyping accuracy statistics to embryonic cell is as follows:
Note：It is the site of heterozygosis before and after " sketch heterozygosis " expression reconstruct；" corrigendum heterozygosis " represents before reconstruct to be homozygosis, is the site of heterozygosis after reconstruct.
5th, β-ground is poor and HLA types judge
A) by father and mother's haplotype, genotype of the embryonic cell on rs7480526 is successfully reduced, it is the poor feminine gender in β-ground to predict it, and finds that the allele with propositus on the position is different, so that it is determined that embryo is the poor feminine gender in β-ground.
B) can be drawn by this method, propositus and embryo in the haplotype in MHC areas as so that the HLA types for judging them are matchings.
According to the poor genotype in β-ground and HLA genotyping results of above-mentioned embryo, so as to select correct embryo, i.e., the embryo that the poor negative and HLA types in β-ground are matched with propositus carries out Cryopreservation and implantation uterus, so as to prevent the gestation of ill youngster.Embodiment 2
First, sample collection and processing：
A couple is the poor carrier in β-ground, receives IVF-PGD treatments.By an IVF-PGD treatment cycle, 8 blastomere embryos were obtained at the 3rd day, the unicellular biopsy sampling of blastomere embryos is carried out, the blastomere after biopsy continued to cultivate to the 5th day Nang embryonic stage, carried out Nang embryo vitrificated cryopreserrations.The unicellular sample of 8 blastomeres that biopsy is obtained carries out whole genome amplification, whole genome amplification uses the REPLI-g MDA amplification kits of Qiagen companies, strictly operated according to kit specification, amplified production is used for new-generation sequencing and other analyses.
The peripheral blood of father, mother in this family are also collected simultaneously, extract genomic DNA. IVF-PGD treatments are completed, are being selected after correct Embryonic limb bud cell uterus, successful pregnancy, amniotic fluid of pregnant woman can be extracted after 14 weeks, or are collecting embryo's Cord blood, DNA is being extracted, is sequenced, to verify the genotyping result of embryonic cell.
The unicellular whole genome amplification product DNA of 8 embryos, father, mother's peripheral blood DNA, and amniotic fluid of pregnant woman or embryo's Cord blood DNA interrupt instrument with Covaris and interrupted to the fragment of 200bp sizes, upper according to illumina companies HiSeq2000 sequenators confidential asks progress to build storehouse, library carries out the capture of All in One chips, and concrete operations are shown in that storehouse specification (http is built in the capture of NimbleGen SeqCap EZ chips://www.nimblegen.com/products/seqcap/ez/index.html can be obtained).All in One chips are the target area capture chips of autonomous Design, and its target area includes full exon region, 1M SNP site region and mhc gene area.Library after chip capture carries out Quality Control through Agilent Bioanalyzer 2100 and Q-PCR methods, qualified library proceeds illumina HiSeq2000 sequencers, and sequencing strategy is the index of Pair End 90 (i.e. two-way 90bp index sequencings）, sequencing depth is the 30-50X of target area.The parameter setting and operating method of its Instrumental all (can be by http according to illumina operation manuals://www.illumina.com/support/documentation.ilmn is obtained).
2nd, sequencing data is analyzed：
1) parent gene group DNA, amniotic fluid DNA (checking sample）Sequencing data is analyzed and parting：
A) sequence of low quality value and joint pollution is filtered out
B) using Burrows-Wheeler Aligner, (BWA, http/bio-bwa.sourceforge.net/W take high shield amount by force：| sequence sequence alignment is in mankind's reference gene group（Hgl9, NCBI release GRCh37).And the sequence of PCR repeat amplification protcols is got rid of with SAMtools.
C) using Genome Analysis Toolkit (GATK, http：〃 www.broadinstitute.org/gatk/) calibrate base mass value and carry out the detection that single base nucleotide polymorphisms and missing are repeated.
2) analyzed according to the unicellular sequencing data of 8 embryos, the genotype information of each embryonic cell genome initialization is provided respectively, build the hereditary sketch of cellular genome of each embryo.
3) any one unicellular embryo to be measured of 8 embryos is determined, according to the hereditary sketch of embryonic cell genome to be measured and the genotype of father and mother, the haplotype of father and mother is initialized.
4) by HMM, the haplotype of father and mother is reconstructed and determined according to the genotype of remaining 7 embryo respectively.
5) by HMM, according to father and mother's haplotype, to reconstruct the haplotype of embryo to be measured.
6) determination of embryo genetic variation information to be measured.
By taking No. 22 chromosome as an example, with reference to amniotic fluid genotypic results, the Genotyping accuracy statistics to embryo to be measured is as follows：Type (female X father）Total number of sites SNP Call rate accuracy rate (heterozygosis）Accuracy rate (homozygosis）
Homozygosis X heterozygosis 2,183 97.16% 96.68% 99.63%
Heterozygosis X homozygosis 2,156 93.18% 98.95% 100%
Heterozygosis X heterozygosis 1,154 82.32% 99.31% 97.59%
7) β-ground is poor and HLA types judge
A) by father and mother's haplotype, haplotype of the embryonic cell to be measured on rs7480526 is successfully reduced, it is the poor feminine gender in β-ground to predict it. B) by this method, with reference to the Genetic Detection result of first disease person, it can determine that whether haplotype of the propositus with embryo in MHC areas be consistent, so as to judge whether their HLA types match.
According to the poor genotype in β-ground and HLA genotyping results of above-mentioned embryo, so as to select correct embryo, i.e., the embryo that the poor negative and HLA types in β-ground are matched with first disease person carries out Cryopreservation and implantation uterus, so as to prevent the gestation of ill youngster.Industrial applicibility
Method, the system and computer-readable medium for determining presumptive area base information in embryonic gene group of presumptive area base information, can be effectively applied to and the nucleotide sequence of presumptive area in embryonic gene group is analyzed in the determination embryonic gene group of the present invention.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 (1)

  1. Claims
    1st, a kind of method for determining presumptive area base information in embryonic gene group, it is characterised in that comprise the following steps：Obtain embryonic cell genomic DNA sample sequencing result, and embryo genetic related individuals genome sample sequencing result；
    Based on the sequencing result of embryonic cell genomic DNA sample, the hereditary sketch of the embryo is built, to determine embryo's initial gene type；
    Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, determine the base information of presumptive area in embryonic gene group.
    2nd, according to the method described in claim 1, it is characterised in that the embryonic gene group DNA sample is obtained from micro embryonic cell, wherein, the micro embryonic cell comes from blastomere, Nang embryos or embryonated egg.
    3rd, method according to claim 2, it is characterised in that the embryonic gene group DNA sample is obtained from embryo is unicellular.
    4th, the method according to claim 1, characterized in that, the sequencing result is to utilize at least one acquisition selected from Illumina-Solexa/Hiseq, ABI-SOLiD, Life Technologies-Ion Torrent/Proton, Roche-454 and single-molecule sequencing device.
    5th, according to the method described in claim 1, it is characterised in that by the way that the sequencing result of embryonic cell genomic DNA sample is compared with reference sequences, the hereditary sketch of the embryo is built.
    6th, according to the method described in claim 1, it is characterised in that by the way that the sequencing result of the embryo genetic related individuals genome sample is compared with reference sequences, determine the genotype of the embryo genetic related individuals；And
    Based on the genotype of the embryo genetic related individuals, the haplotype of the embryo father and mother is determined.
    7th, the method according to claim 5 or 6, it is characterised in that the reference sequences are mankind's reference gene group.
    8th, according to the method described in claim 1, it is characterised in that the embryo genetic related individuals include the father and mother and propositus of the embryo.
    9th, method according to claim 8, it is characterised in that the propositus is other children of the embryo father and mother.
    10th, method according to claim 9, it is characterised in that according to hidden Markov model, determines that the base information of presumptive area in embryonic gene group further comprises：
    Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
    11st, method according to claim 10, it is characterised in that the hidden Markov model uses following parameters：Initial state probabilities are distributed as==0.5), l-NrlNp i = j eS), wherein,Nr 1 NP
    Nr, Np represent to expect respectively restructuring number and mononucleotide polymorphism site number, and Nr is natural number, span 20-40, and observation sequence probability matrix iss ={) }, G'eS, eV), wherein,
    #sites(L > 0,Must-hom.)
    #w'to(L>0, Must-het. or Must-horn.) b;(l) = 1— b;(0)
    #sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation；
    P # L ^ ( J ) = max - 1 ( ) · aij] -bj(K) / F fl Νλ
    Office's Shao's probability is ieil-Wj, and reverse pointer is te{l...N} ^
    qN* =argmaxSN(qN)
    Recursive call end-state is to recall optimal path, and the base information for determining most probable embryo's presumptive area is=ψ"ι(^+1)
    (=1,2,3 ..., N_1)
    12nd, the method according to claim 1, it is characterised in that further comprise：The presumptive area is the known site that there is genetic polymorphism.
    13rd, method according to claim 12, it is characterised in that the genetic polymorphism is at least one selected from SNP and STR.
    14th, a kind of system for determining presumptive area base information in embryonic gene group, it is characterised in that including：Library construction device, the library construction device is suitable to the genomic DNA sample for embryonic gene group DNA sample and embryo genetic related individuals, and sequencing library is built respectively；
    Sequencing device, the sequencing device is connected with the library construction device, and suitable for the sequencing library is sequenced, to obtain the sequencing result of embryo and embryo genetic related individuals；And
    Analytical equipment, the analytical equipment is connected with the sequencing device, and is suitable to：
    Based on the sequencing result of embryonic gene group DNA sample, the hereditary sketch of the embryo is built, to determine embryo's initial gene type；
    Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And
    According to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, the base information of presumptive area in embryonic gene group is determined.
    15th, system according to claim 14, it is characterised in that further comprise DNA sample separator, the DNA sample separator is suitable to carry out biopsy sampling, acquisition amount embryonic cell, from the micro embryonic cell from embryo Embryonic gene group DNA sample is extracted, wherein, the micro embryonic cell comes from blastomere, Nang embryos or embryonated egg.
    16th, the system according to claim 14, characterized in that, the sequencing device is at least one selected from Illumina-Solexa/Hiseq, ABI-SOLiD, Life Technologies-Ion Torrent/Proton, Roche-454 and single-molecule sequencing device.
    17th, system according to claim 14, it is characterised in that further comprise comparison device, the comparison device is connected with the sequencing device, for the sequencing result and reference sequences to be compared, so as to：
    By the way that the sequencing result of embryonic cell genomic DNA sample is compared with reference sequences, the hereditary sketch of the embryo is built；
    By the way that the sequencing result of the embryo genetic related individuals genome sample is compared with reference sequences, the genotype of the embryo genetic related individuals is determined；And
    Based on the genotype of the embryo genetic related individuals, the haplotype of the embryo father and mother is determined.
    18th, system according to claim 14, it is characterised in that according to hidden Markov model, determines that the base information of presumptive area in embryonic gene group further comprises：
    Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
    19th, system according to claim 18, it is characterised in that the hidden Markov model uses following parameters：Initial state probabilities are distributed as==0.5), the probability matrix of l-NrlNp i=j hidden states transfer isk =
    Nr, Np represent to expect respectively restructuring number and mononucleotide polymorphism site number, and Nr is natural number, span 20-40, and observation sequence probability matrix iss =^) }, G'eS, eV), wherein,
    # sites(L > 0,Must-hom.)
    b,(0)
    #w'to(L>0, Must-het. or Must-horn.), (1)=1- b;(0)
    #sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation；
    P # ^ ^ ( J ) = max - 1 ( ) · aij] -bj(K) / F fl Νλ
    Office's Shao's probability is ieil-Wj,
    Ψ,. (_/·) = arg max ― ·α
    Reverse pointer is te { l...N } ^
    qN* =argmaxSN(qN)
    Recursive call end-state is to recall optimal path, and the base information for determining most probable embryo's presumptive area is=ψ ί+1^^ι) (=1,2,3 ..., N_1)
    20th, a kind of computer-readable medium, it is characterised in that be stored with instruction on the computer-readable medium, it is described instruction be suitable to be executed by processor so as to：
    Based on the sequencing result of embryonic cell genomic DNA sample, the hereditary sketch of embryo is built, to determine embryo's initial gene type；
    Based on the sequencing result of the embryo genetic related individuals genome sample, the haplotype of embryo father and mother is determined；And according to hidden Markov model, using embryo's initial gene type as observation sequence, based on the haplotype of the embryo father and mother, determine the base information of presumptive area in embryonic gene group.
    21st, computer media according to claim 20, it is characterised in that by the way that the sequencing result of embryonic cell genomic DNA sample is compared with reference sequences, builds the hereditary sketch of the embryo.
    22nd, computer media according to claim 20, it is characterised in that by the way that the sequencing result of the embryo genetic related individuals genome sample is compared with reference sequences, determine the genotype of the embryo genetic related individuals；And the genotype based on the embryo genetic related individuals, determine the haplotype of the embryo father and mother.
    23rd, computer media according to claim 20, it is characterised in that according to hidden Markov model, determines that the base information of presumptive area in embryonic gene group further comprises：
    Build initial state probabilities distribution vector, the probability matrix and observation sequence probability matrix of hidden state transfer；End-state is determined than algorithm using Hui Te and recall optimal path, to determine the base information of presumptive area in embryonic gene group.
    24th, according to claim 2 in the hidden Markov model uses following parameters：Initial state probabilities are distributed as The probability matrix of l-NrlNp i=j hidden states transfer isA = Wherein,
    Nr, Np represent to expect respectively restructuring number and mononucleotide polymorphism site number, and Nr is natural number, span 20-40, and observation sequence probability matrix iss ={) }, G'eS, eV), wherein,
    #sites(L > 0,Must-hom.)
    #w'to(L>0, Must-het. or Must-horn.) b;(l) = 1— b;(0)
    #sites (L>0, Must-hom.) it is set to the number in the site of homozygosis, #sites (L for filial generation one>0, Must-horn, or Must-het.) for filial generation one be set to homozygosis site number and filial generation one be set to heterozygosis site number summation；
    P # L ^ ( J ) = max - 1 ( ) · aij] -bj(K) / F fl Νλ
    Office's Shao's probability is ieil-Wj, and reverse pointer is te{l...N} ^
    qN* =argmaxSN(qN)
    Recursive call end-state is, Recall optimal path, the base information for determining most probable embryo's presumptive area isq' =Ψ^(^ί + 1) (i = l,2,3,...,N- 1)
    25th, computer-readable medium according to claim 20, it is characterised in that the presumptive area is the known site that there is genetic polymorphism.
    26th, computer-readable medium according to claim 25, it is characterised in that the genetic polymorphism is at least one selected from SNP and STR.
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