HK1208889A1

HK1208889A1 - Method and system for determining whether individual is in abnormal state

Info

Publication number: HK1208889A1
Application number: HK15109589.1A
Authority: HK
Inventors: 王威; 殷旭阳; 张春雷; 陈盛培; 潘小瑜; 张春生
Original assignee: 深圳华大基因股份有限公司
Priority date: 2012-08-23
Filing date: 2012-08-23
Publication date: 2016-03-18
Also published as: WO2014029093A1; CN104508141A

Abstract

Provided are methods and systems for determining whether an individual has an abnormal state. The method for determining whether an individual has an abnormal state includes: constructing a sequencing library based on the individual's nucleic acid sample; Sequencing the sequencing library to obtain sequencing results, which are composed of multiple sequencing data; Based on the sequencing data, determine the known SNPs included in the sequencing results; And based on the known SNP, determine whether the individual has an abnormal state associated with the known SNP.

Description

It is determined that whether individual suffers from the method and system of abnormality

Priority information

Nothing

Technical field

The present invention relates to biomedical sector, the method and system of abnormality whether are suffered from particular to determination individual.

Background technology

Mendelian inheritance disease is also known as single-gene disorder（Herein, Mendelian inheritance disease and single-gene disorder can be with used interchangeablies）, autosomal dominant, autosomal recessive, sex-linkage are dominant, sex-linked recessive inheritance is sick etc. can be divided into according to mode of inheritance.End in May, 2012, the single-gene disorder related gene one included in online mankind's Mendelian inheritance database (OMIM) has 21,250 kinds, wherein disease phenotype describes accurate and its Genetic Mechanisms relatively clear about 3,500 kinds or so.

However, the research to single-gene disorder still has much room for improvement at present.

The content of the invention

It is contemplated that at least solving one of technical problem present in prior art.Therefore, the present invention proposes the method and system whether a kind of determination individual suffers from abnormality.

In one aspect of the invention, the present invention propose it is a kind of determine individual whether suffer from abnormality method.Embodiments in accordance with the present invention, this method includes：For the individual sample of nucleic acid, sequencing library is built；The sequencing library is sequenced, to obtain sequencing result, the sequencing result is made up of multiple sequencing datas；Based on the sequencing data, the known SNP included in the sequencing result is determined;And based on the known SNP, determine whether the individual suffers from the abnormality related to the known SNP.Utilize this method, can be effectively by the way that the SNP included in determination sample of nucleic acid be sequenced, and because these SNP to abnormality are related, thus, it is possible to effectively determine whether the source individual of sample of nucleic acid suffers from the abnormality related to these SNP.

Embodiments in accordance with the present invention, the method whether determination individual suffers from abnormality can also have following additional technical feature：

In one embodiment of the invention, the individual is people.Thus, it is possible to using determination individual according to embodiments of the present invention whether the method for suffering from abnormality, human sample is detected, so as to effectively whether be predicted to people with some abnormalities.

In one embodiment of the invention, the abnormality is disease.It is preferred that, the disease is single-gene disorder.It is just blunt According to the specific example of the present invention, the single-gene disorder is at least one selected from thalassemia and Red Blood Cells Glucose -6- Phosphate dehydrogenase deficiency diseases.In one embodiment of the invention, the thalassemia is β-thalassemia.Thus, it is possible to effectively to the risk of human diseases especially single-gene disorder effectively predict and assess.

In one embodiment of the invention, the sample of nucleic acid is at least a portion of the complete genome DNA of individual.Thus, it is possible to further improve the efficiency and accuracy for determining the method whether individual suffers from abnormality.

In one embodiment of the invention, the sample of nucleic acid is extracted from the unicellular or trace sample of individual.In one embodiment of the invention, described unicellular or trace sample is to be selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one separation of embryo from individual.Thus, it is possible to by whether carrying out effectively predicting and assessing with abnormality to individual to a small amount of sample from individual, so as to improve the efficiency for determining whether individual suffers from abnormality, reduce the cost for determining whether individual suffers from abnormality.Furthermore it is possible to easily obtain these samples from organism, and different samples can be taken specifically to some diseases, so as to take specific analysis means for some special diseases.

In one embodiment of the invention, it is described it is unicellular be by selected from dilution method, mouth suction pipe partition method, micromanipulation, micro-dissections, fluidic cell exclusion, stream control at least one separation.Thereby, it is possible to effectively easily obtain the unicellular of biological specimen, to implement subsequent operation, thus, it is possible to effectively be separated from individual unicellular, follow-up the efficiency whether individual suffers from abnormality is determined so as to further increase.

In one embodiment of the invention, for the individual sample of nucleic acid, build sequencing library and further comprise：The sample of nucleic acid is expanded, to obtain sample of nucleic acid amplified production；And for the sample of nucleic acid amplified production, build the sequencing library.Thus, it is possible to the efficiency for building sequencing library be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

In one embodiment of the invention, the sample of nucleic acid is the complete genome DNA of the unicellular extraction from individual, can be for example the unicellular full base genomic DNA discharged by cracking individual, wherein, it is by least one progress selected from PEP-PCR, DOP-PCR, OmniPlex WGA and MDA to carry out amplification for the complete genome DNA.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

In one embodiment of the invention, for the sample of nucleic acid amplified production, build before the sequencing library, further comprise：The sample of nucleic acid amplified production is screened using nucleic acid probe, to obtain the sample of nucleic acid amplified production from presumptive area；And for the sample of nucleic acid amplified production from presumptive area, build the sequencing library.In one embodiment of the invention, the presumptive area is at least one exon region.Thus, it is possible to the known SNP that presumptive area is included effectively be determined, thus, it is possible to the known SNP according to included in presumptive area such as exon region be effectively improved, to determine the efficiency and accuracy of the abnormality relevant with these SNP. In one embodiment of the invention, the nucleic acid probe is provided in the form of chip.Thus, it is possible to the efficiency screened using nucleic acid probe further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

In one embodiment of the invention, the sequencing be using be selected from Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing systems, Ion Torrents Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS technologies and nano-pore sequencing technology at least one progress.The characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, further increase the efficiency for determining whether individual suffers from abnormality.In one embodiment of the invention, the sequencing is carried out using Illumina Hiseq2000, and the length of the sequencing data is 90bp.Thus, it is possible to the efficiency of follow-up progress snp analysis further be improved, so as to improve the efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, it is described to be based on the sequencing data, the known SNP included in the sequencing result is determined, is by the way that the sequencing data and reference gene are compared into progress.In one embodiment of the invention, the reference gene is known human genome sequence.In one embodiment of the invention, the comparison is that SOAP/SOAP2 softwares are carried out.Thus, it is possible to effectively analyze the sequencing data included in sequencing result, so as to effectively determine the SNP included in sequencing result, and then can improve to determine the efficiency whether individual suffers from abnormality.

In one embodiment of the invention, further comprise filtering the known SNP included in the sequencing result, the filtering is based on following filter condition：SNP calling mass values are more than 20;SNP site sequencing depth is more than 8;SNP sites depth is less than 5 times of genome mean depth；SNP site copy number is not more than 2;It is more than 5 with the distance between SNP site and other nearest SNP sites.Thus, it is possible to effectively be filtered to resulting SNP results, so as to effectively improve the accuracy and efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, the known SNP is located at human chromosome HBB gene area.In one embodiment of the invention, listen and state known SNP for selected from following at least one: rs33985472、 rs63750954、 rs63751128、 rs33978907、 rs34029390、 rs34809925、 rs33953406、 rs33910569、 rs33910569、 rs33971634、 rs3392539 rs33946267、 rs35485099、 rs36015961、 rs33930977、 rs35256489、 rs33952266、 rs33952266、 rs33952266、 rs33914668、 rs33914668、 rs33913413, rs33913413, rs34483965、 rs34793594、 rs35703285、 rs63750433、 rs34690599、 rs63751175、 rs35328027、 rsl609812、 rs34451549、 rs7480526、 rsl0768683、 rs35099082、 rs63750283、 rs63750283、 rs33945777、 rs33945777、 rs33945777、 rs33933298, rs33913712、 rs33995148、 rs33931779、 rs33969400、 rs33922842、 rs 11549407、 rs33974936、 rs33991059、 rs33982568、 rs33948578、 rsll35071、 rs3394300 rs3394300 rs63750513、 rs34527846、 rs35456885、 rs35004220、 rs63750195、 rs35724775、 rs33915217、 rs33915217、 rs33915217、 rs33956879、 rs33956879、 rs33956879、 rs33971440、 rs33971440、 rs33960103、 rs33960103、 rs35684407、 rs35578002、 rs33916412、 rs35424040、 rs33950507、 rs33950507、 rs33951465、 rs33959855、 rs33972047、 rs33986703、 Rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628, rs34305195 and 5246716 on human chromosome 11,5246879,5247026, the SNP of 5248161.Thus, it is possible to effectively determine whether human body carries the SNP in HBB gene area, so as to effectively determine whether institute's research object suffers from the risk of thalassemia especially β-thalassemia.

In another aspect of this invention, the present invention propose it is a kind of determine individual whether suffer from abnormality system.Embodiments in accordance with the present invention, the system includes：Sequencing library construction device, the sequencing library construction device is used to be directed to the individual sample of nucleic acid, builds sequencing library；Sequencing device, the sequencing device is connected with the sequencing library construction device, for the sequencing library to be sequenced, and to obtain sequencing result, the sequencing result is made up of multiple sequencing datas；SNP determining devices, the SNP determining devices are connected with the sequencing device, for based on the sequencing data, determining the known SNP included in the sequencing result;And abnormality determining device, the abnormal device determining device is connected with the SNP determining devices, for based on the known SNP, determining whether the individual suffers from the abnormality related to the known SNP.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality, so as to effectively by the way that the SNP included in determination sample of nucleic acid is sequenced, and because these SNP to abnormality are related, thus, it is possible to effectively determine whether the source individual of sample of nucleic acid suffers from the abnormality related to these SNP.

Embodiments in accordance with the present invention, whether the system with abnormality can also have following additional technical feature to determination individual：

In one embodiment of the invention, the system further comprises：Sample of nucleic acid extraction element, the sample of nucleic acid extraction element is suitable at least a portion that the complete genome DNA of individual is extracted from the unicellular or trace sample of individual.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.

In one embodiment of the invention, the system further comprises：Biological specimen separator, the biological specimen separator is suitable to separate unicellular or trace sample from individual selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one of embryo.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thus, it is possible to by whether carrying out effectively predicting and assessing with abnormality to individual to a small amount of sample from individual, so as to improve the efficiency for determining whether individual suffers from abnormality, reduce the cost for determining whether individual suffers from abnormality.Furthermore it is possible to easily obtain these samples from organism, and different samples can be taken specifically to some diseases, so as to take specific analysis means for some special diseases.

In one embodiment of the invention, the biological specimen separator is suitable to by being separated selected from dilution method, mouth suction pipe Method, micromanipulation, micro-dissections, fluidic cell exclusion, micro-fluidic at least one separate unicellular or trace sample.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thereby, it is possible to effectively easily obtain the unicellular of biological specimen, to implement subsequent operation, thus, it is possible to effectively be separated from individual unicellular, follow-up the efficiency whether individual suffers from abnormality is determined so as to further increase.

In one embodiment of the invention, the sequencing library construction device further comprises：Sample of nucleic acid amplification unit, the sample of nucleic acid amplification unit is suitable to expand the sample of nucleic acid, to obtain sample of nucleic acid amplified production.In one embodiment of the invention, the amplification unit is adapted at least one selected from PEP-PCR, DOP-PCR, OmniPlex WGA and MDA.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

In one embodiment of the invention, the sequencing library construction device further comprises：Nucleic acid probe is provided with Return menus member, the screening unit, to be screened using the nucleic acid probe to the sample of nucleic acid amplified production, to obtain the sample of nucleic acid amplified production from presumptive area；And for the sample of nucleic acid amplified production from presumptive area, build the sequencing library.Thus, it is possible to the known SNP that presumptive area is included effectively be determined, thus, it is possible to the known SNP according to included in presumptive area such as exon region be effectively improved, to determine the efficiency and accuracy of the abnormality relevant with these SNP.

In one embodiment of the invention, the nucleic acid probe is provided in the form of chip.Thus, it is possible to the efficiency screened using nucleic acid probe further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

In one embodiment of the invention, the sequencing device be selected from Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing systems, Ion Torrent, Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS sequencing devices and nano-pore sequencing device at least one.The characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, further increase the efficiency for determining whether individual suffers from abnormality.In one embodiment of the invention, the sequencing is carried out using Illumina Hiseq2000, and the length of the sequencing data is 90bp.Thus, it is possible to the efficiency of follow-up progress snp analysis further be improved, so as to improve the efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, the SNP determining devices further comprise：Comparing unit, the comparing unit is used for by the way that the sequencing data and reference gene are compared into the known SNP determined included in the sequencing result.In one embodiment of the invention, the comparing unit is suitable to be compared using SOAP/SOAP2 softwares.Thus, it is possible to effectively analyze the sequencing data included in sequencing result, so as to effectively determine included in sequencing result SNP, and then can improve determine individual whether suffer from abnormality efficiency.

In one embodiment of the invention, the SNP determining devices further comprise：SNP filter elements, the SNP filter elements are suitable to be based on following filter condition, and the known SNP included in the sequencing result is filtered：SNP calling mass values are more than 20;SNP site sequencing depth is more than 8;SNP site depth is less than 5 times of genome mean depth；SNP site copy number is not more than 2;It is more than 5 with the distance between SNP site and other nearest SNP sites.Thus, it is possible to effectively be filtered to resulting SNP results, so as to effectively improve the accuracy and efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, the known SNP is located at human chromosome HBB gene area.In one embodiment of the invention, the known SNP is selected from following at least one: rs33985472, rs63750954, rs63751128, rs33978907, rs34029390, rs34809925, rs33953406, rs33910569, rs33910569, rs33971634, rs3392539 rs33946267, rs35485099, rs36015961, rs33930977, rs35256489, rs33952266, rs33952266, rs33952266, rs33914668, rs33914668, rs33913413 , rs33913413 , rs34483965, rs34793594, rs35703285, rs63750433, rs34690599, rs63751175, rs35328027, rsl609812, rs34451549, rs7480526, rsl0768683, i rs35099082, rs63750283, rs63750283, rs33945777, rs33945777, rs33945777, rs33933298, rs33913712, rs33995148, rs33931779, rs33969400, rs33922842, rs 11549407, rs33974936, rs33991059, rs33982568, rs33948578, rsl l35071, rs3394300 rs3394300 rs63750513, rs34527846, rs35456885, rs35004220, rs63750195, rs35724775, rs33915217, rs33915217, rs33915217, rs33956879, rs33956879, rs33956879, rs33971440, rs33971440, rs33960103, rs33960103, rs35684407, rs35578002, rs33916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828 , rs63750628 ,Rs34305195 and 5246716 on human chromosome 11,5246879,5247026,The SNP of 5248161.Thus, using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.So as to effectively determine whether institute's research object suffers from the risk of thalassemia especially β-thalassemia.

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 according to an embodiment of the invention for determining the individual method for whether suffering from abnormality；Fig. 2 is the schematic flow sheet in accordance with another embodiment of the present invention for determining the individual method for whether suffering from abnormality；Fig. 3 is the schematic flow sheet for the method that abnormality whether is suffered from according to the determination individual of another embodiment of the invention；Fig. 4 is the structural representation according to an embodiment of the invention for determining the individual system for whether suffering from abnormality；Fig. 5 is the structural representation in accordance with another embodiment of the present invention for determining the individual system for whether suffering from abnormality；And

Fig. 6 is the schematic flow sheet in accordance with another embodiment of the present invention for determining the individual method for whether suffering from abnormality.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.

Term " abnormality " used in herein should broadly understood, and it can be any state different from the normal condition of individual such as people, such as can include disease, dysimmunity.In one embodiment of the invention, the abnormality is disease.Embodiments in accordance with the present invention, the type of disease is not particularly restricted, according to a preferred embodiment of the invention, and the disease is single-gene disorder.Single-gene disorder list base is typically the disease or pathology character controlled by a pair of alleles.Therefore, it can effectively determine whether studied object suffers from the disease by detecting the SNP related to single-gene disorder.Embodiments in accordance with the present invention, can also realize that whether being easy to the risk with related abnormality such as disease to individual is predicted and assesses.According to the specific example of the present invention, the single-gene disorder is at least one selected from thalassemia and Red Blood Cells Glucose -6- Phosphate dehydrogenase deficiency diseases.In one embodiment of the invention, the thalassemia is β-thalassemia.Thus, it is possible to effectively determine the mankind whether with related disease especially single-gene disorder.It is determined that individual whether suffer from abnormality method

In one aspect of the invention, the present invention propose it is a kind of determine individual whether suffer from abnormality method.With reference to Fig. 1, embodiments in accordance with the present invention, this method includes：

S100:For the sample of nucleic acid of individual, sequencing library is built

In this step, the sample of nucleic acid first to individual, builds sequencing library, so as to for follow-up sequencing and interpretation of result.In the present invention, the term that is used " individual, implication be not particularly restricted, can be any organism containing hereditary information, for example, can be people.Thus, it is possible to using determination individual according to embodiments of the present invention whether the method for suffering from abnormality, human sample is detected, so as to effectively whether be predicted to people with some abnormalities.

With reference to Fig. 2, embodiments in accordance with the present invention can further include the step of extracting sample of nucleic acid from individual

(S101) used term " sample of nucleic acid " should broadly understood herein, and it can be DNA sample, also may be used To be that DNA sample or RNA samples are modified or handled to obtain to RNA samples or process, its genetic sequence is determined as long as can pass through to be sequenced.According to one embodiment of present invention, sample of nucleic acid can be at least a portion of the complete genome DNA of individual.Thus, whole hereditary information of individual are contained in complete genome DNA, thus, by complete genome DNA being sequenced and snp analysis, individual SNP information more effectively can be intactly obtained, so as to further improve the efficiency and accuracy that determine the method whether individual suffers from abnormality.In one embodiment of the invention, the sample of nucleic acid is extracted from the unicellular or trace sample of individual.Embodiments in accordance with the present invention, the ways and means that sample of nucleic acid is obtained from unicellular or trace sample is not particularly restricted, and single celled complete genome DNA is discharged and collect for example, can realize slender cellular lysate by using lysate.In one embodiment of the invention, described unicellular or trace sample is to be selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one separation of embryo from individual.Thus, it is possible to by whether carrying out effectively predicting and assessing with abnormality to individual to a small amount of sample from individual, so as to improve the efficiency for determining whether individual suffers from abnormality, reduce the cost for determining whether individual suffers from abnormality.Furthermore it is possible to easily obtain these samples from organism, and different samples can be taken specifically to some diseases, so as to take specific analysis means for some special diseases.In one embodiment of the invention, it is described it is unicellular be by selected from dilution method, mouth suction pipe partition method, micromanipulation, micro-dissections, fluidic cell exclusion, micro-fluidic at least one separation.Thereby, it is possible to effectively easily obtain the unicellular of biological specimen, to implement subsequent operation, thus, it is possible to effectively be separated from individual unicellular, follow-up the efficiency whether individual suffers from abnormality is determined so as to further increase.

In addition, embodiments in accordance with the present invention, after sample of nucleic acid is obtained, can be expanded to resulting sample of nucleic acid, especially for the sample of nucleic acid from the extraction of unicellular or trace sample.For example, with reference to Fig. 3, in one embodiment of the invention, for the individual sample of nucleic acid, build sequencing library and further comprise：Resulting sample of nucleic acid is expanded, to obtain sample of nucleic acid amplified production（S102 ).Next, sequencing library can be built to resulting sample of nucleic acid amplified production.Thus, it is possible to the efficiency for building sequencing library be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

Embodiments in accordance with the present invention, the method expanded to sample of nucleic acid, are not particularly restricted.For example, in one embodiment of the invention, the sample of nucleic acid used is, from the complete genome DNA of the unicellular middle extraction of individual, at least one progress selected from PEP-PCR, DOP-PCR, OmniPlex WGA and MDA can be passed through by carrying out amplification for the certificate genomic DNA.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.Optionally, embodiments in accordance with the present invention, may further include to it is described it is unicellular crack, the step of to discharge the single celled full-length genome.According to some examples of the present invention, it can be used for cracking method that is unicellular and discharging full-length genome and be not particularly limited, as long as preferably can fully crack slender cellular lysate.According to the specific example of the present invention, it is possible to use alkaline bleach liquor cleavage liquid is by the slender cellular lysate and discharges described Single celled full-length genome.Inventor has found, so can effectively crack unicellular and discharge full-length genome, and the full-length genome discharged is when being sequenced, it is possible to increase accuracy rate, so as to further increase the efficiency for determining unicellular chromosomal aneuploidy.Embodiments in accordance with the present invention, the method of unicellular whole genome amplification is not particularly limited, PEP-PCR, DOP-PCR and OmniPlex WGA can for example be used using the method for PCR-based, it would however also be possible to employ be not based on PCR method such as MDA (multiple strand displacement amplifications）.According to the specific example of the present invention, it is preferred to use the method for PCR-based, such as OmniPlex WGA methods.Available commercial kit includes but is not limited to Sigma Aldrich GenomePlex, Rubicon Genomics PicoPlex, Qiagen REPLI-g, GE Healthcare illustra GenomiPhi etc..Thus, according to the specific example of the present invention, before sequencing library is built, unicellular full-length genome can be expanded using OmniPlex WGA.Thereby, it is possible to effectively be expanded to full-length genome, so as to further increase the efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, for the sample of nucleic acid amplified production, build before the sequencing library, further comprise：The sample of nucleic acid amplified production is screened using nucleic acid probe, to obtain the sample of nucleic acid amplified production from presumptive area；And for the sample of nucleic acid amplified production from presumptive area, build the sequencing library.In one embodiment of the invention, the presumptive area is at least one exon region.Thus, it is possible to the known SNP that presumptive area is included effectively be determined, thus, it is possible to the known SNP according to included in presumptive area such as exon region be effectively improved, to determine the efficiency and accuracy of the abnormality relevant with these SNP.In addition, embodiments in accordance with the present invention, the method screened by nucleic acid probe to sample of nucleic acid amplified production is not particularly restricted, can be solid-phase screening or solution hybridization.Embodiments in accordance with the present invention, can provide nucleic acid probe in the form of chip.Thus, it is possible to the efficiency screened using nucleic acid probe further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.

Skilled artisans appreciate that be, the sample of nucleic acid of presumptive area can also be analyzed by other known method, PC is carried out to sample of nucleic acid for example with specific primer, thus, the related amplified production of presumptive area can be obtained, so as to build the sequencing library of the presumptive area, and then obtain the relevant information of the presumptive area.

Embodiments in accordance with the present invention, the method to the sample of nucleic acid structure sequencing library of individual is not particularly restricted.Those skilled in the art can select the different methods for building genome sequencing library according to the concrete scheme of the genomic sequencing technique of use, details on building genome sequencing library, 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#1005361;Feb 2010) or Paired-End SamplePrep Guide (Part#1005063;Feb 2010), by referring to be incorporated into herein.

S200:Sequencing library is sequenced, sequencing result is obtained

In this step, by the way that above constructed sequencing library is sequenced, it can obtain by multiple sequencing datas（ reads ) The sequencing result of composition.

Embodiments in accordance with the present invention, technology and platform for sequencing are not particularly restricted.In one embodiment of the invention, it is possible to use be sequenced selected from Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing systems, Ion Torrents Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS technologies and at least one of nano-pore sequencing technology.The characteristics of thereby, it is possible to using the high flux of these sequencing devices, deep sequencing, further increase the efficiency for determining whether individual suffers from abnormality.Certainly, it will be appreciated to those of skill in the art that genome sequencing, such as third generation sequencing technologies, and the more advanced sequencing technologies that may be developed can also be carried out using other sequence measurements and device later.Embodiments in accordance with the present invention, are not particularly limited by the length of the sequencing data obtained by genome sequencing.According to the specific example of the present invention, carried out using Illumina Hiseq2000, the length of the sequencing data is 90bp.Applicant have surprisingly discovered that, when the length of sequencing data is about 90bp, it can greatly facilitate and sequencing data is analyzed, analysis efficiency be improved, while the cost of analysis can be significantly reduced.Further increase and determine whether individual suffers from the efficiency of abnormality, and reduce the cost for determining whether individual suffers from abnormality.Term " sequencing data " used herein above refers to the average value of each sequencing data length value.

S300:Determine the known SNP included in sequencing result

After sequencing result is obtained, it can obtain the hereditary information included in sequencing result by analyzing the sequencing data included in sequencing result, such as, can obtain SNP information.

Embodiments in accordance with the present invention, in this step, to the sequencing data included in sequencing result analyze and obtain the method for SNP information and be not particularly restricted.Embodiments in accordance with the present invention, can be by the way that resulting sequencing data and reference gene be compared, so as to the SNP information in the sequencing result obtained by determining.Those skilled in the art can determine the reference gene for being compared according to analysis purpose.Embodiments in accordance with the present invention, the reference gene used is known human genome sequence, for example can be Hgl9, NCBI Build 37 are certain, those skilled in the art can also be for example compared by using other known sequence as canonical sequence using known SNP as canonical sequence.

Embodiments in accordance with the present invention, are compared used method and software is not particularly restricted.Embodiments in accordance with the present invention, in one embodiment of the invention, can carry out the comparison between sequencing data and canonical sequence using SOAP/SOAP2 softwares.Embodiments in accordance with the present invention, can also be assembled to sequencing data first, and assembling result is compared with canonical sequence.Thus, it is possible to effectively analyze the sequencing data included in sequencing result, so as to effectively determine the SNP included in sequencing result, and then can improve to determine the efficiency whether individual suffers from abnormality.

It will be appreciated by persons skilled in the art that for different microarray datasets, different comparison software can be used.The short sequencing data data of Illumina sequencing systems can be carried out precise alignment by such as SOAP/SOAP2 softwares.Specifically, Quality Control can be carried out to sequencing data according to information such as comparison result Quality of Statistical Data value, comparison rate, G/C content, repetitive rate, genome coverage, sequencing depth, and according to information above.Hash that is can not comparing or repeating is removed simultaneously, Effective data set is obtained, for subsequent analysis.

In addition, in one embodiment of the invention, after SNP information by comparison, is obtained, resulting SNP information can also be screened, for example can by based on filter condition set in advance to being filtered to the known SNP included in sequencing result.Embodiments in accordance with the present invention, the filter condition that can be used is at least one following：

SNP calling mass values are more than 20, and used term " SNP calling mass values " refers in SOAP analysis software runnings herein, to the marking result given by each SNP confidence level.

SNP site sequencing depth is more than 8;

SNP site depth is less than 5 times of genome mean depth；

SNP site copy number is not more than 2;With

The distance between SNP site and other nearest SNP sites are more than 5, expression way the distance between " two SNP site " used in herein refers to the base being separated by between the base number being separated by between the two SNP sites, such as " distance is more than 5 " i.e. two sites more than 5.

Thus, it is possible to effectively be filtered to resulting SNP results, so as to effectively improve the accuracy and efficiency for determining whether individual suffers from abnormality.

S400:It is determined that the abnormality relevant with known SNP

As it was previously stated, obtaining sequencing data by sequencing, and after analyzing sequencing data, the SNP information included in sequencing result can be obtained.And then, can be by analyzing SNP, it is determined that whether individual suffers from the abnormality related to SNP, for example whether with the single-gene disorder related to SNP.

Embodiments in accordance with the present invention, hand is not limited especially the SNP types that can be used.In one embodiment of the invention, the known SNP is located at human chromosome HBB gene area.In one embodiment of the invention, the known SNP is selected from following at least one: rs33985472、 rs63750954、 rs63751128、 rs33978907、 rs34029390、 rs34809925、 rs33953406、 rs33910569、 rs33910569、 rs33971634、 rs3392539 rs33946267、 rs35485099、 rs36015961、 rs33930977、 rs35256489、 rs33952266、 rs33952266、 rs33952266、 rs33914668、 rs33914668、 rs33913413 , rs33913413 , rs34483965、 rs34793594、 rs35703285、 rs63750433、 rs34690599、 rs63751175、 rs35328027、 rsl609812、 rs34451549、 rs7480526、 rsl0768683、 rs35099082、 rs63750283、 rs63750283、 rs33945777、 rs33945777、 rs33945777、 rs33933298, rs33913712、 rs33995148、 rs33931779、 rs33969400、 rs33922842、 rsl 1549407、 rs33974936、 rs33991059、 rs33982568、 rs33948578、 rsl l3507 rs3394300 rs3394300 rs63750513、 rs34527846、 rs35456885、 rs35004220、 rs63750195、 rs35724775、 rs33915217、 rs33915217、 rs33915217、 rs33956879、 rs33956879、 rs33956879、 rs33971440、 rs33971440、 rs33960103、 rs33960103、 rs35684407、 rs35578002、 rs33916412、 rs35424040、 rs33950507、 Rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs34716011, rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628, rs34305195 and 5246716 on human chromosome 11, 5246879, 5247026, the SNP of 5248161.Thus, it is possible to effectively determine whether human body carries the SNP in HBB gene area, so as to effectively determine whether institute's research object suffers from thalassemia especially β-thalassemia.

Thus, the SNP information of individual can be effectively obtained by the above method, and required SNP information can be extracted for the related gene of studied specified disease, the genotyping result of target gene is obtained.After the genotyping result to the target gene of acquisition is obtained, can be by being compared with existing database, disease annotation is carried out, judges whether corresponding SNP variations information can cause disease, and judges the corresponding embryo of this unicellular or trace sample or individual for Mendelian inheritance disease（The disease of single-gene one）Patient, or corresponding SNP variations information are unlikely to cause disease, judge the carrier of corresponding embryo or individual for Mendelian inheritance ospc gene.

It is determined that individual whether suffer from abnormality system

In another aspect of this invention, with reference to Fig. 4, the present invention proposes the system 1000 whether a kind of determination individual suffers from abnormality.Just blunt according to embodiments of the invention, the system 1000 includes：Sequencing library construction device 100, sequencing device 200,

SNP determining devices 300 and abnormality determining device 400.

Just blunt according to embodiments of the invention, sequencing library construction device 100 is used for the sample of nucleic acid for individual, builds sequencing library.Just blunt according to embodiments of the invention, sequencing device 200 is connected with sequencing library construction device 100, thus, it is possible to for constructed sequencing library to be sequenced, to obtain the sequencing result being made up of multiple sequencing datas.Embodiments in accordance with the present invention, SNP determining devices 300 are connected with the sequencing device, for based on resulting sequencing data, determining the known SNP included in sequencing result.Embodiments in accordance with the present invention, abnormality determining device 400 is connected with SNP determining devices, thus, for based on the above identified known SNP included in sequencing result, it is determined that whether individual suffers from the abnormality related to the known SNP.

Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality so that effectively to be determined by being sequenced included in sample of nucleic acid

SNP, and because these SNP to abnormality are related, thus, it is possible to effectively determine whether the source individual of sample of nucleic acid suffers from the abnormality related to these SNP.

In one embodiment of the invention, the system can further include sample of nucleic acid extraction element 101.Embodiments in accordance with the present invention, sample of nucleic acid extraction element 101 is suitable at least a portion that the complete genome DNA of individual is extracted from the unicellular or trace sample of individual.In one embodiment of the invention, the system 1000 may further include suitable for from individual selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one of embryo separation it is unicellular or The biological specimen separator of trace sample.Thus, using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thus, it is possible to by whether carrying out effectively predicting and assessing with abnormality to individual to a small amount of sample from individual, so as to improve the efficiency for determining whether individual suffers from abnormality, reduce the cost for determining whether individual suffers from abnormality.Furthermore it is possible to easily obtain these samples from organism, and different samples can be taken specifically to some diseases, so as to take specific analysis means for some special diseases.In one embodiment of the invention, biological specimen separator is suitable to by separating unicellular or trace sample selected from dilution method, mouth suction pipe partition method, micromanipulation, micro-dissections, fluidic cell exclusion, micro-fluidic at least one.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thereby, it is possible to effectively easily obtain the unicellular of biological specimen, to implement subsequent operation, thus, it is possible to effectively be separated from individual unicellular, follow-up the efficiency whether individual suffers from abnormality is determined so as to further increase.In one embodiment of the invention, sequencing library construction device may further include sample of nucleic acid amplification unit, and the sample of nucleic acid amplification unit is suitable to expand the sample of nucleic acid, to obtain sample of nucleic acid amplified production.In one embodiment of the invention, the amplification unit is adapted at least one selected from PEP-PCR, DOP-PCR, OmniPlex WGA and MDA.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.Using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is foregoing determine individual whether the method for suffering from abnormality.Thus, it is possible to the efficiency of amplification complete genome DNA further be improved, so as to further increase the efficiency whether follow-up determination individual suffers from abnormality.Herein, the method of operation of " sample of nucleic acid extraction element " is not particularly restricted, as long as the sample of nucleic acid that can be obtained the sample of nucleic acid of correlation and be obtained is suitable to subsequent operation, for example for that from unicellular or trace sample complete genome DNA, can be realized by using lysate by slender cellular lysate and discharge and collect single celled complete genome DNA.

In addition, in one embodiment of the invention, sequencing library construction device can further include screening unit.Nucleic acid probe is provided with the screening unit, to be screened using nucleic acid probe to the sample of nucleic acid amplified production, to obtain the sample of nucleic acid amplified production from presumptive area；And for the sample of nucleic acid amplified production from presumptive area, build the sequencing library.Thus, it is possible to the known SNP that presumptive area is included effectively be determined, thus, it is possible to the known SNP according to included in presumptive area such as exon region be effectively improved, to determine the efficiency and accuracy of the abnormality relevant with these SNP.In one embodiment of the invention, nucleic acid probe can be provided in the form of chip.Thus, the efficiency screened using nucleic acid probe can further be improved, so as to further increase it is follow-up determine individual whether the efficiency with abnormality in one embodiment of the invention, the sequencing device be selected from Illumina Hiseq2000, Genome Analyzer ^ SOLiD sequencing systems, Ion Torrent, Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS sequencing devices and nano-pore sequencing device at least one.Thereby, it is possible to utilize the height of these sequencing devices The characteristics of flux, deep sequencing, further increase the efficiency for determining whether individual suffers from abnormality.In one embodiment of the invention, the sequencing is carried out using Illumina Hiseq2000, and the length of the sequencing data is 90bp.Thus, it is possible to the efficiency of follow-up progress snp analysis further be improved, so as to improve the efficiency for determining whether individual suffers from abnormality.In one embodiment of the invention, SNP determining devices further comprise：Comparing unit, the comparing unit is used for by the way that the sequencing data and reference gene are compared into the known SNP determined included in the sequencing result.In one embodiment of the invention, the comparing unit is suitable to be compared using SOAP/SOAP2 softwares.Thus, it is possible to effectively analyze the sequencing data included in sequencing result, so as to effectively determine the SNP included in sequencing result, and then can improve to determine the efficiency whether individual suffers from abnormality.

In one embodiment of the invention, SNP determining devices may further include：SNP filter elements, the SNP filter elements are suitable to be based on following filter condition, and the known SNP included in the sequencing result is filtered：SNP calling mass values are more than 20;SNP site sequencing depth is more than 8;SNP site depth is less than 5 times of genome mean depth；SNP site copy number is not more than 2;It is more than 5 with the distance between SNP site and other nearest SNP sites.Thus, it is possible to effectively be filtered to resulting SNP results, so as to effectively improve the accuracy and efficiency for determining whether individual suffers from abnormality.

In one embodiment of the invention, the known SNP is located at human chromosome HBB gene area.In one embodiment of the invention, the known SNP is selected from following at least one: rs33985472, rs63750954, rs63751128, rs33978907, rs34029390, rs34809925, rs33953406, rs33910569, rs33910569, rs33971634, rs3392539 rs33946267, rs35485099, rs36015961, rs33930977, rs35256489, rs33952266, rs33952266, rs33952266, rs33914668, rs33914668, rs33913413 , rs33913413 , rs34483965, rs34793594, rs35703285, rs63750433, rs34690599, rs63751175, rs35328027, rsl609812, rs34451549, rs7480526, rsl0768683, i rs35099082, rs63750283, rs63750283, rs33945777, rs33945777, rs33945777, rs33933298, rs33913712, rs33995148, rs33931779, rs33969400, rs33922842, rs 11549407, rs33974936, rs33991059, rs33982568, rs33948578, rsl l35071, rs3394300 rs3394300 rs63750513, rs34527846, rs35456885, rs35004220, rs63750195, rs35724775, rs33915217, rs33915217, rs33915217, rs33956879, rs33956879, rs33956879, rs33971440, rs33971440, rs33960103, rs33960103, rs35684407, rs35578002, rs33916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828 , rs63750628 ,Rs34305195 and 5246716 on human chromosome 11,5246879,5247026,The SNP of 5248161.Thus, using determination individual according to embodiments of the present invention whether the system with abnormality can effectively implement it is noted earlier really Fixed individual whether the method for suffering from abnormality.So as to effectively judge whether institute's research object suffers from thalassemia especially β-thalassemia.

Term used herein above " is connected;; it should broadly understood; both can be to be joined directly together; can also be indirectly connected to; it is even possible that with identical container or equipment, as long as linking functionally can be realized, such as sample of nucleic acid is extracted and sample of nucleic acid amplification can be carried out in the same equipment, i.e. after realizing to sample of nucleic acid extraction, sample of nucleic acid amplification processing can be carried out in identical equipment or container, it is not necessary to which the sample of nucleic acid group extracted is delivered to other equipment or container, as long as by the condition in equipment（Composition including reaction condition and reaction system）Be converted to and be adapted for sample of nucleic acid amplified reaction, so realize sample of nucleic acid extract with sample of nucleic acid amplification being connected functionally, it is also assumed that being covered by term " connected ".

Skilled artisans appreciate that be, it is previously with regard to determine the feature and advantage whether individual is suffered from described by the method for abnormality, also of course using language the present invention determination individual whether suffer from abnormality system, for convenience, repeat no more.

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 described by document in the art or condition (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

As non-limiting example, in a particular embodiment of the present invention, with reference to Fig. 6, using the method comprised the following steps, single-gene disorder is carried out to unicellular or trace sample and detected：

S1 :Unicellular separation/trace sample prepares；

S2:Whole genome amplification；

S3:Sample is sequenced and prepares (library preparation)；

S4:High-flux sequence；

S5:Comparing and Quality Control, obtain valid data collection；

S6: SNP Calling;

S7:SNP is extracted and filtered, and obtains the genotyping result of target gene；

S8:Disease is annotated.

Embodiment 1

Sample：People's 5-8 cell stage blastomeres are unicellular

Concrete operations flow： 1. the unicellular separation of people's 5-8 cell stages blastomere

Fertilization-embryo PGD in vitro（IVF-PGD) in periodic process, sperm and ovum after fertilization in vitro, in vitro culture formed the blastomere of 5-8 cell stages to the 3rd day.Routine autopsy was carried out at the 3rd day, under micromanipulation instrument, one blastomere of taking-up is unicellular, is placed in the PCR pipe containing lysate, -80 °C of preservations.Blastomere after biopsy continued to cultivate to the 5th day, reaches Nang embryonic stages, can carry out glass freezing, or be directly used in implantation.

2. whole genome amplification

From the REPLI-g Mini Kit kits of Qiagen companies, the code provided according to manufacturer carries out unicellular whole genome amplification, blastomere is unicellular to carry out alkaline lysis first, then adds amplification reaction solution and carries out 30 °C of constant-temperature amplifications.

3. sample, which is sequenced, prepares (library construction）

Using Illumina Paired-End DNA Sample Prep Kit, the code provided according to manufacturer, constructed dna sequencing library.For above resulting, the unicellular whole genome amplification product of blastomere carries out building storehouse, three libraries is prepared for altogether, it is respectively 200bp, 350bp, 500bp that Insert Fragment is expected in library, is actually inserted into clip size and is shown in Table 1.

4. high-flux sequence

High-flux sequence is carried out using Illumina Hiseq2000 sequencing systems.To the strategy of the unicellular use genome sequencing of blastomere in the present embodiment.The library that the unicellular amplified production of blastomere is prepared prepares Cluster through cBot, afterwards i.e. in the operation of Hiseq2000 sequenators, and the two-way sequencings of length 90bp, Pair End are sequenced, and a lane is surveyed in each library ,-three lane have been surveyed altogether.

5. comparing and Quality Control, obtain valid data collection

Sequencing data is compared with SOAP2 softwares, and genome sequence (Hgl9 is referred to people;NCBI Build37) to refer to, the mispairing of most two bases is allowed during comparison.Raw data quality, G/C content are counted according to comparison result, clip size, comparison rate, repetitive rate is actually inserted into, and genome the information such as coverage and sequencing depth.Specifying information is shown in Table 1.And Quality Control is carried out to sequencing data by these statistical results.In the present embodiment, we obtain full-length genome 38.25X data volume altogether, and data items statistical result can be up to standard.The data for failing to compare the data of upper reference gene group and repeat are removed afterwards, valid data collection are obtained, for snp analysis.

The comparison statistical result * of the unicellular sequencing data of blastomere in the embodiment 1 of table 1

Sample P E reads PE Unique Coverage Mean Depth Duplication Rate

BLSl 392.08M 382.26M (97.50%) 91.34% 14.21 0.70%

BLSl 368.13M 359.19M (97.57%) 91.71% 13.45 1.04%

BLSl 357.44M 348.37M (97.46%) 89.93% 12.95 0.58%

BLSl

1.12G 1.09G (97.51%) 97.04% 38.25 0.77% (total)

Mark：

* BLSl is the numbering of the unicellular sample of blastomere, BLSl (total) represent BLSl three lane data of sample be combined after statistical result, Q20 (%) represents that data of the mass value more than 20 account for the ratio of total amount of data, GC (%) represents the actual G/C content percentage of sequencing data, Insert Size represent the actual library inserts size of sequencing data, Clean Reads represent remove low quality value read after remaining read data volumes, PE-alignment represents that Pair End two ends can compare the read data volumes of reference gene group and account for total amount of data ratio, PE reads represent that Pair End two ends can compare the read data volumes of reference gene group, PE Unique represent that Pair End two ends can uniquely compare the read data volumes of reference gene group, Coverage represents the coverage of full-length genome, Mean Depth represent the mean depth of full-length genome, Duplication Rate represent that repeating read data accounts for total read data volumes ratio.

6. SNP Calling

The present embodiment SOAPsnp softwares, according to SOAPsnp operation instruction（With reference to webpage http://soa genomics.org.cn/soapsn, html, by referring to being incorporated into herein) SNP Calling are carried out to the valid data collection of above-mentioned acquisition, finally obtain SNP data sets.

7. SNP is extracted and filtered, the genotyping result of target gene is obtained

The SNP data sets of above-mentioned acquisition are filtered, filter condition is as follows：

A. SNP calling mass values are more than 20;

B. sequencing depth in the site is more than 8;

C. the site depth is less than 5 times of genome mean depth；

D. the site copy number is not more than 2;

E. the distance between the SNP and nearest SNP is more than 5.

SNP data after filtering, for the disease related gene to be detected, extract the SNP being located in target gene area.The gene of beta Thalassemia disease is detected in the present embodiment, to detect whether corresponding blastomere embryos sick with beta Thalassemia, or whether be beta Thalassemia disease gene carrier.Therefore the present embodiment is extracted the SNP site positioned at No. 11 chromosome Η Β Β gene regions, and specifying information is shown in Table 2.

The SNP site information * positioned at Η Β Β gene regions obtained in the embodiment 1 of table 2

The Please Ο Ζ OAV of/ZT0ZN3/X3d ￡ 606

The Please Ο Ζ OAV of/ZT0ZN3/X3d ￡ 606 chrl l 5248249 C CC C->A rs33930702 HBB chrl l 5248250 A AA A->G rs33941849 HBB chrl l 5248250 A AA A->C rs33941849 HBB chrl l 5248250 A AA A->T rs33941849 HBB chrl l 5248251 T TT T->C rs34563000 HBB chrl l 5248269 G GG G->C rs34135787 HBB chrl l 5248280 C CC C->T rs34704828 HBB chrl l 5248282 G GG G->A rs63750628 HBB chrl l 5248301 T TT T->G rs34305195 HBB are marked：

^Chromosome represents chromosome number, Locus represents the site numbering of the corresponding bases of SNP on chromosome, Ref represents base type of the people with reference to correspondence site on genome in database, Blastomere represents the type information of correspondence SNP site in the unicellular data of blastomere, Mutation represents the mutation type in correspondence site present in database, SNP ID represent ID numbering of the SNP site in database, and Gene represents which gene regions is the SNP site be located at.

8. disease is annotated

According to above-mentioned filtering and the SNP information of the target gene of extraction, disease annotation is further done.In the present embodiment, it is found that heterozygosis SNP makes a variation respectively in 5247141 and 5247791 bit bases of No. 11 chromosomes, they are respectively positioned on HBB gene area.The homozygous mutation in the two sites can cause beta Thalassemia disease.And the site of the two in the present embodiment is heterozygous mutant, illustrate the carrier that the unicellular corresponding blastomere is beta Thalassemia Disease-causing gene.

Embodiment 2

Sample：Normal human blood is unicellular

Concrete operations flow：

1. the unicellular separation of normal human blood

The present embodiment blood sample comes from a normal individual human of phenotype.A small amount of blood sample is extracted, through centrifugation, leukocytic cream is isolated.After leucocyte is washed through PBS, it is suspended in PBS droplets, is separated mononuclear leukocyte with mouth suction pipe, is placed in the alkaline cell lysis liquid of 1-2 μ 1, -80 °C freeze more than 30min.

2. whole genome amplification

From the REPLI-g Mini Kit kits of Qiagen companies, the code provided according to manufacturer, blood mononuclear cell adds amplification reaction solution and carries out 30 °C of constant-temperature amplifications after 65 °C of processing in 10 minutes carry out alkaline lysis.

3. chip captures library construction

Blood mononuclear cell sample whole genome amplification product carries out chip capture specific target areas, and carries out library construction simultaneously.The target area of Agilent chip pins pair used is altogether 2.1M sizes in the present embodiment, includes all exon regions of 100 kinds of single-gene disorder related genes.On the application method of Agilent chips, the code that manufacturer is provided may be referred to. High-flux sequence will be carried out by the sequencing library for capturing and building.

4. high-flux sequence

The present embodiment carries out high-flux sequence using Illumina Hiseq2000 sequencing systems.Capture the strategy of sequencing in the present embodiment using chip to blood mononuclear cell, target area is all exon regions of 100 kinds of single-gene disorder related genes, about 2.1M sizes.The chip capture library that blood mononuclear cell amplified production is prepared prepares Cluster through cBot, the two-way sequencings of length 90bp, Pair End is sequenced in the operation of Hiseq2000 sequenators afterwards, sequencing data amount is contemplated to 1 ~ 2G bases.

5. comparing and Quality Control, obtain valid data collection

Sequencing data is compared with SOAP2 softwares, and genome sequence (Hgl9 is referred to people;NCBI Build37) to refer to, the mispairing of most two bases is allowed during comparison.The information such as coverage and sequencing depth according to comparison result statistics Raw data quality, G/C content, comparison rate, repetitive rate, and genome.Specifying information is shown in Table 3. and carries out Quality Control to sequencing data by these statistical results.In the present embodiment, we obtain target area mean depth 457X data volume altogether, and data items statistical result can be up to standard.The data for failing to compare the data of upper reference gene group and repeat are removed afterwards, valid data collection are obtained, for snp analysis.

The comparison statistical result of blood mononuclear cell sequencing data in the embodiment 2 of table 3

Mark：

* GC (%) represents the actual G/C content percentage of sequencing data, Reads represent remove low quality value read after remaining read data volumes, Production represents the base data volume calculated according to Reads values, PE-alignment represents that Pair End two ends can compare the read data volumes of reference gene group and account for total amount of data ratio, PE Unique represent that Pair End two ends can uniquely compare the read data volumes of reference gene group, Coverage represents the coverage of full-length genome, Mean Depth represent the mean depth of full-length genome, Duplication Rate represent that repeating read data accounts for total read data volumes ratio, Specificity (Reads) represents that the read data volumes that can specifically compare target area account for the ratio of total read data volumes, Specificity (Bases) represents that the base data volume that can specifically compare target area accounts for ratio of the total bases according to amount.

6. SNP Calling

The present embodiment SOAPsnp softwares, according to SOAPsnp operation instruction（With reference to webpage http://soap.genoniics, org m''soapsnp.htrnl, by referring to be incorporated into herein) to the valid data collection of above-mentioned acquisition SNP Calling are carried out, SNP data sets are finally obtained.

A. SNP calling mass values are more than 20;

B. sequencing depth in the site is more than 8;

C. the site depth is less than 5 times of target area mean depth；

D. the site copy number is not more than 2;

E. the distance between the SNP and nearest SNP is more than 5.

SNP data after filtering, for the disease related gene to be detected, extract the SNP being located in target gene area.The gene of beta Thalassemia disease is detected in the present embodiment, to detect whether corresponding blastomere embryos sick with beta Thalassemia, or whether be beta Thalassemia disease gene carrier.Therefore the present embodiment is extracted the SNP site positioned at No. 11 chromosome Η Β Β gene regions, and specifying information is shown in Table 4.

It is located at the SNP site information of Η Β Β gene regions in the embodiment 2 of table 4

The Please Ο Ζ OAV of 0/ZT0ZN3/X3d ￡ 606 chrl l 5248158 A AA A->T rs33956879 HBB chrl l 5248159 C CC C->T rs33971440 HBB chrl l 5248159 C CC C->A rs33971440 HBB chrl l 5248160 C CC C->T rs33960103 HBB chrl l 5248160 C CC C->G rs33960103 HBB chrl 1 5248161 T TT T->G unknown HBB chrl 1 5248161 T TT T->C rs35684407 HBB chrl l 5248162 G GG G->A rs35578002 HBB chrl l 5248166 A AA A->C rs33916412 HBB chrl l 5248170 C CC C->A rs35424040 HBB chrl l 5248173 C CC C->T rs33950507 HBB chrl l 5248173 C CC C->A rs33950507 HBB chrl l 5248177 A AA A->T rs33951465 HBB chrl l 5248185 C CC C->A rs33959855 HBB chrl 1 5248193 T TT T->C rs33972047 HBB chrl 1 5248200 T TT T->A rs33986703 HBB chrl l 5248204 C CC C->T rs34716011 HBB chrl l 5248205 C CC C->T rs63750783 HBB chrl l 5248219 G GG G->T rs35799536 HBB chrl l 5248249 C CC C->T rs33930702 HBB chrl l 5248249 C CC C->G rs33930702 HBB chrl l 5248249 C CC C->A rs33930702 HBB chrl l 5248250 A AA A->G rs33941849 HBB chrl l 5248250 A AA A->C rs33941849 HBB chrl l 5248250 A AA A->T rs33941849 HBB chrl 1 5248251 T TT T->C rs34563000 HBB chrl l 5248269 G GG G->C rs34135787 HBB chrl l 5248280 C CC C->T rs34704828 HBB chrl l 5248282 G GG G->A rs63750628 HBB chrl 1 5248301 T TT T->G rs34305195 HBB are marked：

* Chromosome represents chromosome number, Locus represents the site numbering of the corresponding bases of SNP on chromosome, Ref represents base type of the people with reference to correspondence site on genome in database, Blood Cell represent the type information of correspondence SNP site in blood mononuclear cell data, Mutation represents the mutation type in correspondence site present in database, SNP ID represent ID numbering of the SNP site in database, and Gene represents which gene regions is the SNP site be located at.

8. disease is annotated

The SNP information of the target gene of above-mentioned filtering and extraction, further does disease annotation.In the present embodiment; the variant sites of any heterozygosis or homozygosis are not detected in HBB gene regions; illustrate that the corresponding individual of the blood mononuclear cell in the present embodiment is not beta Thalassemia Disease; nor beta Thalassemia disease gene carrier, its beta Thalassemia disease gene area is normal genotype. So far, embodiments of the invention realize a kind of sick to unicellular or trace sample detection Mendelian inheritance based on high-flux sequence（Single-gene disorder）Method.

Industrial applicibility

The present invention's, structure and the sequencing in the DNA sequencing library of sample DNA can be effectively applied to, and the Library Quality obtained is good, and sequencing result is accurate.

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

1st, it is a kind of determine individual whether suffer from abnormality method, it is characterised in that including：

For the individual sample of nucleic acid, sequencing library is built；

The sequencing library is sequenced, to obtain sequencing result, the sequencing result is made up of multiple sequencing datas；Based on the sequencing data, the known SNP included in the sequencing result is determined;And

Based on the known SNP, determine whether the individual suffers from the abnormality related to the known SNP.

2nd, according to the method described in claim 1, it is characterised in that the individual is people.

3rd, according to the method described in claim 1, it is characterised in that the abnormality is disease.

4th, method according to claim 3, it is characterised in that the disease is single-gene disorder.

5th, method according to claim 4, it is characterised in that the single-gene disorder is at least one selected from thalassemia and Red Blood Cells Glucose -6- Phosphate dehydrogenase deficiency diseases.

6th, method according to claim 5, it is characterised in that the thalassemia is β-thalassemia.

7th, the method according to claim 1, it is characterised in that the sample of nucleic acid is at least a portion of the complete genome DNA of individual.

8th, according to the method described in claim 1, it is characterised in that the sample of nucleic acid is extracted from the unicellular or trace sample of individual.

9th, method according to claim 8, it is characterised in that described unicellular or trace sample is to be selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one separation of embryo from individual.

10th, the method according to claim 9, it is characterised in that it is described it is unicellular be by selected from dilution method, mouth suction pipe partition method, aobvious operation, aobvious cutting, fluidic cell exclusion, micro-fluidic at least one separation.

11st, the method according to claim 1, it is characterised in that for the individual sample of nucleic acid, builds sequencing library and further comprises：

The sample of nucleic acid is expanded, to obtain sample of nucleic acid amplified production；And

For the sample of nucleic acid amplified production, the sequencing library is built.

12nd, method according to claim 11, it is characterised in that the sample of nucleic acid is the complete genome DNA of the unicellular middle extraction from individual,

Its towel,

It is by least one progress selected from PEP-PCR, DOP-PCR, OmniPlex WGA and MDA to carry out amplification for the complete genome DNA.

13rd, method according to claim 11, it is characterised in that for the sample of nucleic acid amplified production, build before the sequencing library, further comprise： The sample of nucleic acid amplified production is screened using nucleic acid probe, to obtain the sample of nucleic acid amplified production from presumptive area；And

For the sample of nucleic acid amplified production from presumptive area, the sequencing library is built.

14th, method according to claim 13, it is characterised in that the nucleic acid probe is provided in the form of chip.15th, method according to claim 13, it is characterised in that the presumptive area is at least one exon region.

16th, according to the method described in claim 1, characterized in that, the sequencing be using be selected from Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing systems, Ion Torrent, Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS technologies and nano-pore sequencing technology at least one progress.

17th, method according to claim 16, it is characterised in that the sequencing is carried out using Illumina Hiseq2000, the length of the sequencing data is 90bp.

18th, the method according to claim 1, it is characterised in that described to be based on the sequencing data, determines the known SNP included in the sequencing result, is by the way that the sequencing data and reference gene are compared into progress.

19th, method according to claim 18, it is characterised in that the reference gene is known human genome sequence.

20th, method according to claim 18, it is characterised in that the comparison is that SOAP/SOAP2 softwares are carried out.21st, method according to claim 20, it is characterised in that further comprise filtering the known SNP included in the sequencing result, the filtering is based on following filter condition：

SNP calling mass values are more than 20;

SNP site sequencing depth is more than 8;

SNP site depth is less than 5 times of genome mean depth；

SNP site copy number is not more than 2;With

The distance between SNP site and other nearest SNP sites are more than 5.

22nd, according to the method described in claim 1, it is characterised in that the known SNP is located at human chromosome HBB gene area.

23rd, method according to claim 22, it is characterised in that, the known SNP is selected from following at least one： rs33985472、 rs63750954、 rs63751128、 rs33978907、 rs34029390、 rs34809925、 rs33953406、 rs33910569、 rs33910569、 rs33971634、 rs3392539 rs33946267、 rs35485099、 rs36015961、 rs33930977、 rs35256489、 rs33952266、 rs33952266、 rs33952266、 rs33914668、 rs33914668、 rs33913413, rs33913413, rs34483965、 rs34793594、 rs35703285、 rs63750433、 rs34690599、 rs63751175、 rs35328027、 rsl609812、 rs34451549、 rs7480526、 rs 10768683、 rs35099082、 rs63750283、 rs63750283、 rs33945777、 rs33945777、 rs33945777、 rs33933298, rs33913712、 rs33995148、 rs33931779、 rs33969400、 rs33922842、 rsl 1549407、 rs33974936、 rs33991059、 rs33982568, rs33948578, rsl l3507 rs3394300 rs3394300 rs63750513, rs34527846, rs35456885, rs35004220, rs63750195, rs35724775, rs33915217, rs33915217, rs33915217, rs33956879, rs33956879, rs33956879, rs33971440, rs33971440, rs33960103, rs33960103, rs35684407, rs35578002, rs33916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628,Rs34305195 and 5246716 on human chromosome 11,5246879,5247026,The SNP of 5248161.

24th, it is a kind of determine individual whether suffer from abnormality system, it is characterised in that including：

Sequencing library construction device, the sequencing library construction device is used to be directed to the individual sample of nucleic acid, builds sequencing library；

Sequencing device, the sequencing device is connected with the sequencing library construction device, for the sequencing library to be sequenced, and to obtain sequencing result, the sequencing result is made up of multiple sequencing datas；

SNP determining devices, the SNP determining devices are connected with the sequencing device, for based on the sequencing data, determining the known SNP included in the sequencing result;And

Abnormality determining device, the abnormal device determining device is connected with the SNP determining devices, for based on the known SNP, determining whether the individual suffers from the abnormality related to the known SNP.

25th, system according to claim 24, it is characterised in that further comprise：

Sample of nucleic acid extraction element, the sample of nucleic acid extraction element is suitable at least a portion that the complete genome DNA of individual is extracted from the unicellular or trace sample of individual.

26th, system according to claim 25, it is characterised in that further comprise：

Biological specimen separator, the biological specimen separator is suitable to separate unicellular or amount sample from individual selected from blood, tissue, urine, gamete, embryonated egg, blastomere and at least one of embryo.

27th, system according to claim 26, characterized in that, the biological specimen separator is suitable to separate unicellular or trace sample by least one selected from dilution method, mouth suction pipe partition method, aobvious operation, aobvious cutting, fluidic cell exclusion, stream control.

28th, system according to claim 24, it is characterised in that the sequencing library construction device further comprises：Sample of nucleic acid amplification unit, the sample of nucleic acid amplification unit is suitable to expand the sample of nucleic acid, to obtain sample of nucleic acid amplified production.

29th, system according to claim 24, it is characterised in that the amplification unit be adapted for selected from PEP-PCR,

DOP-PCR, OmniPlex WGA and MDA at least one. 30th, system according to claim 29, it is characterised in that the sequencing library construction device further comprises：Nucleic acid probe is provided with screening unit, Suo Shu Return menus member, to be screened using the nucleic acid probe to the sample of nucleic acid amplified production, to obtain the sample of nucleic acid amplified production from presumptive area；And

For the sample of nucleic acid amplified production from presumptive area, the sequencing library is built.

31st, system according to claim 30, it is characterised in that the nucleic acid probe is provided in the form of chip.

32nd, system according to claim 24, characterized in that, the sequencing device be selected from Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing systems, Ion Torrent, Ion Proton, 454, PacBio RS sequencing systems, Helicos tSMS sequencing devices and nano-pore sequencing device at least one.

33rd, system according to claim 24, it is characterised in that the SNP determining devices further comprise：Comparing unit, the comparing unit is used for by the way that the sequencing data and reference gene are compared into the known SNP determined included in the sequencing result.

34th, system according to claim 33, it is characterised in that the comparing unit is suitable to be compared using SOAP/SOAP2 softwares.

35th, system according to claim 33, it is characterised in that the SNP determining devices further comprise：SNP filter elements, the SNP filter elements are suitable to be based on following filter condition, and the known SNP included in the sequencing result is filtered：

SNP calling mass values are more than 20;

SNP site sequencing depth is more than 8;

SNP site depth is less than 5 times of genome mean depth；

SNP site copy number is not more than 2;With

The distance between SNP site and other nearest SNP sites are more than 5.

36th, system according to claim 24, it is characterised in that the known SNP is located at human chromosome HBB gene area.

37th, system according to claim 36, it is characterised in that the known SNP is selected from following at least one: rs33985472、 rs63750954、 rs63751128、 rs33978907、 rs34029390、 rs34809925、 rs33953406、 rs33910569、 rs33910569、 rs33971634、 rs3392539 rs33946267、 rs35485099、 rs36015961、 rs33930977、 rs35256489、 rs33952266、 rs33952266、 rs33952266、 rs33914668、 rs33914668、 rs33913413, rs33913413、 rs34483965、 rs34793594、 rs35703285、 rs63750433、 rs34690599、 rs63751175、 rs35328027、 rsl609812、 rs34451549、 rs7480526、 rsl0768683、 rs35099082、 rs63750283、 rs63750283、 rs33945777、 rs33945777、 rs33945777、 rs33933298、 rs33913712、 rs33995148、 rs33931779、 rs33969400、 rs33922842、 rsll549407、 rs33974936、 rs33991059、 rs33982568, rs33948578, rsl l3507 rs3394300 rs3394300 rs63750513, rs34527846, rs35456885, rs35004220, rs63750195, rs35724775, rs33915217, rs33915217, rs33915217, rs33956879, rs33956879, rs33956879, rs33971440, rs33971440, rs33960103, rs33960103, rs35684407, rs35578002, rs33916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs33941849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628,Rs34305195 and 5246716 on human chromosome 11,5246879,5247026,The SNP of 5248161.