WO2014029093A1

WO2014029093A1 - Method and system for determining whether individual is in abnormal state

Info

Publication number: WO2014029093A1
Application number: PCT/CN2012/080500
Authority: WO
Inventors: 王威; 殷旭阳; 张春雷; 陈盛培; 潘小瑜; 张春生
Original assignee: 深圳华大基因科技有限公司; 深圳华大基因研究院
Priority date: 2012-08-23
Filing date: 2012-08-23
Publication date: 2014-02-27
Also published as: CN104508141A; HK1208889A1

Abstract

Provided are a method and a system for determining whether an individual is in an abnormal state. The method for determining whether the individual is in the abnormal state comprises: establishing a sequencing library for a nucleic acid sample of the individual; sequencing the sequencing library to acquire a sequencing result, wherein the sequencing result is formed by multiple pieces of sequencing data; determining, based on the sequencing data, a known SNP comprised in the sequencing result; and determining, based on the known SNP, whether the individual is in an abnormal state related to the known SNP.

Description

Method and system for determining whether an individual has an abnormal state

Priority information

No

Technical field

The present invention relates to the field of biomedicine and, in particular, to methods and systems for determining whether an individual has an abnormal state.

Background technique

Mendelian genetic disease, also known as single-gene disease (in this paper, Mendelian genetic disease and single-gene disease are used interchangeably), according to genetic methods can be divided into autosomal dominant, autosomal recessive, with sexual dominant , with concealed recessive genetic diseases. As of May 2012, there are 21,250 single-gene disease-related genes already included in the online human Mendelian Genetic Database (OMIM), of which about 3,500 are characterized by accurate disease phenotypes and clear genetic mechanisms. .

However, current research on single-gene diseases remains to be improved.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method and system for determining whether an individual has an abnormal state.

In one aspect of the invention, the invention proposes a method of determining whether an individual has an abnormal state. According to an embodiment of the invention, the method comprises: constructing a sequencing library for the nucleic acid sample of the individual; sequencing the sequencing library to obtain a sequencing result, the sequencing result consisting of a plurality of sequencing data; Sequencing the data, determining a known SNP contained in the sequencing result; and determining whether the individual has an abnormal state associated with the known SNP based on the known SNP. With this method, the SNPs contained in the nucleic acid samples can be efficiently determined by sequencing, and since these SNPs are related to the abnormal state, it is possible to effectively determine whether the source individuals of the nucleic acid samples have abnormalities associated with these SNPs. status.

According to an embodiment of the invention, the method of determining whether an individual has an abnormal state may also have the following additional technical features:

In one embodiment of the invention, the individual is a human. Thus, the human body sample can be detected by the method of determining whether the individual has an abnormal state according to an embodiment of the present invention, so that it is possible to effectively predict whether the person has some abnormal state.

In one embodiment of the invention, the abnormal state is a disease. Preferably, the disease is a monogenic disease. Talent According to a specific example of the present invention, the monogenic disease is at least one selected from the group consisting of thalassemia and erythrocyte glucose-6-phosphate dehydrogenase deficiency. In one embodiment of the invention, the thalassemia is beta-thalassemia. Thereby, the risk of disease of human diseases, especially single-gene diseases, can be effectively predicted and evaluated.

In one embodiment of the invention, the nucleic acid sample is at least a portion of an individual's whole genome DNA. Thereby, the efficiency and accuracy of the method of determining whether an individual has an abnormal state can be further improved.

In one embodiment of the invention, the nucleic acid sample is extracted from a single cell or a microsample of the individual. In one embodiment of the invention, the single or minimal sample is isolated from at least one member selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres, and embryos. Thus, it is possible to effectively predict and evaluate whether an individual has an abnormal state by a small sample from an individual, thereby improving the efficiency of determining whether an individual has an abnormal state, and reducing the cost of determining whether an individual has an abnormal state. In addition, these samples can be easily obtained from organisms and can be specifically sampled for certain diseases to take specific analytical measures for specific diseases.

In one embodiment of the invention, the single cells are isolated by at least one selected from the group consisting of a dilution method, a mouth pipette separation method, micromanipulation, microdissection, flow cytometry, and flow control. Thereby, the single cells of the biological sample can be obtained efficiently and conveniently, so that the subsequent operations can be performed, whereby the single cells can be effectively separated from the individual, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

In one embodiment of the invention, constructing the sequencing library for the nucleic acid sample of the individual further comprises: amplifying the nucleic acid sample to obtain a nucleic acid sample amplification product; and amplifying the product for the nucleic acid sample, The sequencing library was constructed. Thereby, the efficiency of constructing the sequencing library can be improved, thereby further improving the efficiency of subsequent determination of whether or not the individual has an abnormal state.

In one embodiment of the invention, the nucleic acid sample is whole genome DNA extracted from a single cell of an individual, for example, may be a whole-base genomic DNA released by lysing a single cell of an individual, wherein the whole genome DNA is Amplification is carried out by at least one selected from the group consisting of PEP-PCR, DOP-PCR, OmniPlex WGA and MDA. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state.

In one embodiment of the present invention, before constructing the sequencing library for the nucleic acid sample amplification product, the method further comprises: screening the nucleic acid sample amplification product by using a nucleic acid probe to obtain a predetermined region. Amplification product of the nucleic acid sample; and constructing the sequencing library for the nucleic acid sample amplification product from the predetermined region. In an embodiment of the invention, the predetermined area is at least one exon area. Thereby, the known SNPs included in the predetermined area can be effectively determined, whereby the efficiency of determining the abnormal state related to the SNPs according to the predetermined SNPs included in the predetermined area such as the exon area can be effectively improved. And accuracy. In one embodiment of the invention, the nucleic acid probe is provided in the form of a chip. Thereby, the efficiency of screening using the nucleic acid probe can be further improved, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

In one embodiment of the invention, the sequencing is performed using at least one selected from the group consisting of Illumina Hiseq 2000, Genome Analyzer, SOLiD sequencing system, Ion Torrents Ion Proton, 454, PacBio RS sequencing system, Helicos tSMS technology, and nanopore sequencing technology. ongoing. Thereby, the high-throughput, deep-sequencing characteristics of these sequencing devices can be utilized to further improve the efficiency of determining whether an individual has an abnormal state. In one embodiment of the invention, the sequencing is performed using Illumina Hiseq 2000, which is 90 bp in length. Thereby, the efficiency of subsequent SNP analysis can be further improved, thereby improving the efficiency of determining whether an individual has an abnormal state.

In one embodiment of the present invention, the determining the known SNPs included in the sequencing result based on the sequencing data is performed by comparing the sequencing data with a reference gene. In one embodiment of the invention, the reference gene is a known human genomic sequence. In one embodiment of the invention, the alignment is performed by SOAP/SOAP2 software. Thereby, the sequencing data included in the sequencing result can be effectively analyzed, so that the SNP contained in the sequencing result can be effectively determined, and the efficiency of determining whether the individual has an abnormal state can be improved.

In an embodiment of the present invention, the method further comprises filtering the known SNPs included in the sequencing result, the filtering is based on the following filtering conditions: SNP calling quality value is greater than 20; SNP site sequencing depth is greater than 8; SNP The depth of the locus is less than 5 times the average depth of the genome; the copy number of the SNP locus is no more than 2; and the distance between the SNP locus and the nearest other SNP loci is greater than 5. Thereby, the obtained SNP results can be effectively filtered, so that the accuracy and efficiency of determining whether an individual has an abnormal state can be effectively improved.

In one embodiment of the invention, the known SNP is located in the human chromosome HBB gene region. In one embodiment of the invention, the known SNP is said to be at least one selected from the group consisting of: 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 SNPs located on human chromosome 11 at positions 5246716, 5246879, 5247026, 5248161. Thereby, it is possible to effectively determine whether the human body carries the SNP of the HBB gene region, thereby being able to effectively determine whether the subject has a risk of thalassemia, especially β-thalassemia.

In another aspect of the invention, the invention proposes a system for determining whether an individual has an abnormal state. According to an embodiment of the present invention, the system comprises: a sequencing library construction device for constructing a sequencing library for a nucleic acid sample of the individual; a sequencing device, the sequencing device and the sequencing library construction device Connected for sequencing the sequencing library to obtain a sequencing result, the sequencing result consisting of a plurality of sequencing data; a SNP determining device, the SNP determining device being coupled to the sequencing device for performing the sequencing based Data, determining a known SNP included in the sequencing result; and abnormal state determining means, the abnormal device determining means being connected to the SNP determining means for determining whether the individual is suffering based on the known SNP There are abnormal states associated with the known SNPs. A method of determining whether an individual has an abnormal state, as described above, using a system according to an embodiment of the present invention for determining whether an individual has an abnormal state, thereby efficiently determining a SNP contained in a nucleic acid sample by sequencing, and Since these SNPs are related to abnormal states, it is thereby possible to effectively determine whether the source individuals of the nucleic acid samples have abnormal states associated with these SNPs.

According to an embodiment of the invention, the system for determining whether an individual has an abnormal state may also have the following additional technical features:

In one embodiment of the invention, the system further comprises: a nucleic acid sample extraction device adapted to extract at least a portion of the individual's whole genomic DNA from a single cell or a micro sample of the individual. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention.

In one embodiment of the invention, the system further comprises: a biological sample separation device adapted to be selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres, and embryos At least one separates a single cell or a microsample. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thus, it is possible to effectively predict and evaluate whether an individual has an abnormal state by a small sample from an individual, thereby improving the efficiency of determining whether an individual has an abnormal state, and reducing the cost of determining whether an individual has an abnormal state. In addition, these samples can be easily obtained from organisms and can be specifically sampled for certain diseases to take specific analytical tools for specific diseases.

In an embodiment of the invention, the biological sample separation device is adapted to be separated by a method selected from a dilution method and a mouth pipette Method, micromanipulation, microdissection, flow cytometry, microfluidic separation of at least one isolated single cell or microsample. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thereby, the single cells of the biological sample can be obtained efficiently and conveniently, so that the subsequent operations can be performed, whereby the single cells can be effectively separated from the individual, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

In an embodiment of the present invention, the sequencing library construction device further comprises: a nucleic acid sample amplification unit, wherein the nucleic acid sample amplification unit is adapted to amplify the nucleic acid sample to obtain a nucleic acid sample amplification product. In one embodiment of the invention, the amplification unit is adapted to perform at least one selected from the group consisting of PEP-PCR, DOP-PCR, OmniPlex WGA, and MDA. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state.

In an embodiment of the present invention, the sequencing library construction device further includes: a sorting unit, wherein the screening unit is provided with a nucleic acid probe to screen the nucleic acid sample amplification product by using the nucleic acid probe And obtaining a nucleic acid sample amplification product from a predetermined region; and constructing the sequencing library for the nucleic acid sample amplification product from the predetermined region. Thereby, the known SNPs included in the predetermined area can be effectively determined, whereby the efficiency of determining the abnormal state related to the SNPs according to the predetermined SNPs included in the predetermined area such as the exon area can be effectively improved. And accuracy.

In one embodiment of the invention, the nucleic acid probe is provided in the form of a chip. Thereby, the efficiency of screening using the nucleic acid probe can be further improved, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

In one embodiment of the invention, the sequencing device is at least selected from the group consisting of Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing system, Ion Torrent, Ion Proton, 454, PacBio RS sequencing system, Helicos tSMS sequencing device, and nanopore sequencing device. One. Thereby, it is possible to further improve the efficiency of determining whether an individual has an abnormal state by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices. In one embodiment of the invention, the sequencing is performed using Illumina Hiseq 2000, which is 90 bp in length. Thereby, the efficiency of subsequent SNP analysis can be further improved, thereby improving the efficiency of determining whether an individual has an abnormal state.

In an embodiment of the present invention, the SNP determining apparatus further includes: a comparing unit, configured to determine, by comparing the sequencing data with a reference gene, the included in the sequencing result Know the SNP. In one embodiment of the invention, the comparison unit is adapted to perform alignment using SOAP/SOAP2 software. Thereby, the sequencing data included in the sequencing result can be effectively analyzed, thereby effectively determining the inclusion in the sequencing result. The SNP, in turn, can increase the efficiency of determining whether an individual has an abnormal state.

In an embodiment of the present invention, the SNP determining apparatus further includes: a SNP filtering unit, wherein the SNP filtering unit is adapted to filter the known SNPs included in the sequencing result based on the following filtering conditions: SNP calling The mass value is greater than 20; the SNP site sequencing depth is greater than 8; the SNP site depth is less than 5 times the genome average depth; the SNP site copy number is not greater than 2; and the distance between the SNP site and the nearest other SNP site is greater than 5. Thereby, the obtained SNP results can be effectively filtered, so that the accuracy and efficiency of determining whether an individual has an abnormal state can be effectively improved.

In one embodiment of the invention, the known SNP is located in the human chromosome HBB gene region. In one embodiment of the present invention, the known SNP is at least one selected from the group consisting of: 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, rs33 971440, 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 SNPs located on human chromosome 11 at 5246716, 5246879, 5247026, and 5248161. Thus, the above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. It is thus possible to effectively determine whether the subject has a risk of thalassemia, especially beta-thalassemia.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from 1 is a flow chart showing a method of determining whether an individual has an abnormal state according to an embodiment of the present invention; FIG. 2 is a flow chart showing a method of determining whether an individual has an abnormal state according to another embodiment of the present invention; A schematic diagram of a method for determining whether an individual has an abnormal state according to still another embodiment of the present invention; FIG. 4 is a schematic structural view of a system for determining whether an individual has an abnormal state according to an embodiment of the present invention; A structural schematic diagram of another embodiment of a system for determining whether an individual has an abnormal state;

6 is a flow chart showing a method of determining whether an individual has an abnormal state, according to another embodiment of the present invention. Detailed description of the invention

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.

The term "abnormal state" as used herein shall be understood broadly, and may be any state different from the normal state of an individual such as a person, and may include, for example, a disease, an immune abnormality, or the like. In one embodiment of the invention, the abnormal state is a disease. According to an embodiment of the present invention, the type of the disease is not particularly limited, and according to a preferred embodiment of the present invention, the disease is a single gene disease. A single gene disease monotherapy is usually a disease or pathological trait controlled by a pair of alleles. Therefore, by detecting SNPs associated with a single gene disease, it is possible to effectively determine whether the subject under study has the disease. According to an embodiment of the present invention, it is also possible to predict and evaluate whether an individual is prone to suffer from an associated abnormal state such as a disease. According to a specific example of the present invention, the monogenic disease is at least one selected from the group consisting of thalassemia and erythrocyte glucose-6-phosphate dehydrogenase deficiency. In one embodiment of the invention, the thalassemia is beta-thalassemia. Thereby, it is possible to effectively determine whether a human has a related disease, especially a monogenic disease. Method of determining whether an individual has an abnormal state

In one aspect of the invention, the invention proposes a method of determining whether an individual has an abnormal state. Referring to FIG. 1, in accordance with an embodiment of the present invention, the method includes:

S100: Construction of a sequencing library for individual nucleic acid samples

In this step, a sequencing library is first constructed for individual nucleic acid samples, which can be used for subsequent sequencing and results analysis. In the present invention, the term "individual" is used without limitation, and may be any organism containing genetic information, for example, may be human. Thus, it is possible to determine whether an individual is suffering from an embodiment according to an embodiment of the present invention. The method of abnormal state detects the human body sample, so that it can effectively predict whether a person has some abnormal state.

Referring to FIG. 2, according to an embodiment of the present invention, the method further includes the step of extracting a nucleic acid sample from an individual.

(S101) „ The term “nucleic acid sample” as used herein shall be understood broadly and may be a DNA sample or It can be an RNA sample, or it can be a modified or processed DNA sample or RNA sample, as long as the genetic sequence can be determined by sequencing. According to one embodiment of the invention, the nucleic acid sample can be at least a portion of an individual's whole genome DNA. Thus, the whole genome DNA contains all the genetic information of the individual, and thus, by sequencing and SNP analysis of the whole genome DNA, the SNP information of the individual can be obtained more effectively and completely, thereby further improving whether the individual has an abnormality. The efficiency and accuracy of the state method. In one embodiment of the invention, the nucleic acid sample is extracted from a single cell or a microsample of the individual. According to an embodiment of the present invention, the method and means for obtaining a nucleic acid sample from a single cell or a micro sample are not particularly limited, and for example, single cell cleavage may be carried out by using a lysate to effect release and collect single cell whole genome DNA. In one embodiment of the invention, the single or minimal sample is isolated from at least one member selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres, and embryos. Thus, the efficiency of determining whether an individual has an abnormal state can be improved by effectively predicting and evaluating whether an individual has an abnormal state by a small sample from an individual, and the cost of determining whether the individual has an abnormal state is reduced. In addition, these samples can be easily obtained from organisms, and can be specifically sampled for certain diseases to take specific analytical measures for certain specific diseases. In one embodiment of the invention, the single cells are isolated by at least one selected from the group consisting of a dilution method, a mouth pipette separation method, micromanipulation, microdissection, flow cytometry, and microfluidics. Thereby, the single cells of the biological sample can be obtained efficiently and conveniently, so that the subsequent operations can be performed, whereby the single cells can be effectively separated from the individual, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

Further, according to an embodiment of the present invention, after obtaining a nucleic acid sample, the obtained nucleic acid sample can be expanded, especially for a nucleic acid sample extracted from a single cell or a micro sample. For example, referring to Fig. 3, in one embodiment of the present invention, constructing a sequencing library for the nucleic acid sample of the individual further comprises: amplifying the obtained nucleic acid sample to obtain a nucleic acid sample amplification product (S102). Next, a product can be amplified from the obtained nucleic acid sample to construct a sequencing library. Thereby, the efficiency of constructing the sequencing library can be improved, thereby further improving the efficiency of subsequent determination of whether the individual has an abnormal state.

The method of amplifying a nucleic acid sample according to an embodiment of the present invention is not particularly limited. For example, in one embodiment of the invention, the nucleic acid sample employed is whole genome DNA extracted from a single cell of an individual, and amplification of the coupon genomic DNA can be performed by selecting from PEP-PCR, DOP-PCR, OmniPlex Conducted by at least one of WGA and MDA. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state. Optionally, according to an embodiment of the present invention, the step of lysing the single cells to release the whole genome of the single cells may be further included. According to some examples of the present invention, a method which can be used for lysing a single cell and releasing a whole genome is not particularly limited as long as single cell lysis can be preferably sufficiently lysed. According to a specific example of the present invention, the single cell can be cleaved and released using an alkaline lysate Single-genome whole genome. The inventors have found that this can effectively lyse single cells and release the whole genome, and the released whole genome can improve the accuracy when sequencing, thereby further improving the efficiency of determining single cell chromosome aneuploidy. According to an embodiment of the present invention, the method of single-cell whole genome amplification is not particularly limited, and PCR-based methods such as PEP-PCR, DOP-PCR, and OmniPlex WGA may be employed, and non-PCR-based methods may be employed, for example. MDA (multiple strand displacement amplification). According to a specific example of the invention, a PCR based method, such as the OmniPlex WGA method, is preferably employed. Commercial kits of choice include, but are not limited to, GenomePlex from Sigma Aldrich, PicoPlex from Rubicon Genomics, REPLI-g from Qiagen, illustra GenomiPhi from GE Healthcare, and the like. Thus, according to a specific example of the invention, the single cell whole genome can be amplified using OmniPlex WGA prior to construction of the sequencing library. Thereby, the whole genome can be efficiently amplified, thereby further improving the efficiency of determining whether the individual has an abnormal state.

In one embodiment of the present invention, before constructing the sequencing library for the nucleic acid sample amplification product, the method further comprises: screening the nucleic acid sample amplification product by using a nucleic acid probe to obtain a predetermined region. Amplification product of the nucleic acid sample; and constructing the sequencing library for the nucleic acid sample amplification product from the predetermined region. In an embodiment of the invention, the predetermined area is at least one exon area. Thereby, the known SNPs included in the predetermined area can be effectively determined, whereby the efficiency of determining the abnormal state related to the SNPs according to the predetermined SNPs included in the predetermined area such as the exon area can be effectively improved. And accuracy. Further, according to an embodiment of the present invention, the method of screening the amplification product of the nucleic acid sample by means of the nucleic acid probe is not particularly limited, and may be a solid phase screening or a liquid phase hybridization. According to an embodiment of the invention, the nucleic acid probe can be provided in the form of a chip. Thereby, the efficiency of screening using the nucleic acid probe can be further improved, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state.

It will be understood by those skilled in the art that the nucleic acid sample of the predetermined region can also be analyzed by other known methods, for example, the nucleic acid sample is subjected to PC using a specific primer, thereby obtaining a related amplification product of a predetermined region. Thereby, a sequencing library of the predetermined region is constructed, and information about the predetermined region is obtained.

According to an embodiment of the present invention, a method of constructing a sequencing library for a nucleic acid sample of an individual is not particularly limited. Those skilled in the art can select different methods for constructing a whole genome sequencing library according to the specific scheme of the genome sequencing technology adopted. For details on constructing the whole genome sequencing library, refer to the protocol provided by the manufacturer of the sequencing instrument, such as Illumina, for example, see Illumina Corporation Multiplexing Sample Preparation Guide (Part #1005361; Feb 2010) or Paired-End SamplePrep Guide (Part #1005063; Feb 2010), which is incorporated herein by reference.

S200: Sequencing the sequencing library to obtain sequencing results

In this step, by sequencing the previously constructed sequencing library, multiple sequencing data (lists) can be obtained. The resulting sequencing results.

Techniques and platforms for sequencing are not particularly limited in accordance with embodiments of the present invention. In one embodiment of the invention, sequencing can be performed using at least one selected from the group consisting of Illumina Hiseq 2000, Genome Analyzer, SOLiD sequencing system, Ion Torrents Ion Proton, 454, PacBio RS sequencing system, Helicos tSMS technology, and nanopore sequencing technology. Thereby, it is possible to further improve the efficiency of determining whether an individual has an abnormal state by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices. Of course, those skilled in the art will appreciate that other sequencing methods and devices can be used for whole genome sequencing, such as third generation sequencing techniques, as well as more advanced sequencing technologies that may be developed in the future. According to an embodiment of the present invention, the length of the sequencing data obtained by whole genome sequencing is not particularly limited. According to a specific example of the present invention, the sequencing data is 90 bp in length using Illumina Hiseq2000. Applicants have surprisingly found that when the length of the sequencing data is about 90 bp, the sequencing data can be greatly facilitated, the analysis efficiency is improved, and the cost of the analysis can be significantly reduced. The efficiency of determining whether an individual has an abnormal state is further improved, and the cost of determining whether the individual has an abnormal state is reduced. The term "sequence data" as used herein refers to the average of the length values of individual sequencing data.

S300: Determine the known SNPs included in the sequencing results

After obtaining the sequencing result, the genetic information contained in the sequencing result can be obtained by analyzing the sequencing data included in the sequencing result, for example, SNP information can be obtained.

According to an embodiment of the present invention, in this step, the method of analyzing the sequencing data contained in the sequencing result to obtain the SNP information is not particularly limited. According to an embodiment of the present invention, SNP information in the obtained sequencing result can be determined by comparing the obtained sequencing data with a reference gene. Those skilled in the art can determine the reference genes used for the alignment based on the analytical purpose. According to an embodiment of the present invention, the reference gene used is a known human genome sequence, which may be, for example, Hgl9, NCBI Build 37. Of course, those skilled in the art may also use other known sequences as reference sequences, for example, The known SNPs were used as a reference sequence for alignment.

According to an embodiment of the present invention, the method and software employed for the comparison are not particularly limited. In accordance with an embodiment of the present invention, in one embodiment of the invention, the alignment between the sequencing data and the reference sequence can be performed using SOAP/SOAP2 software. According to an embodiment of the present invention, sequencing data may also be assembled first, and the assembly result is compared with a reference sequence. Thereby, the sequencing data contained in the sequencing result can be effectively analyzed, so that the SNP contained in the sequencing result can be effectively determined, and the efficiency of determining whether the individual has an abnormal state can be improved.

Those skilled in the art will appreciate that different alignment software can be employed for different sequencing platforms. For example, SOAP/SOAP2 software provides accurate alignment of short-sequence data from Illumina sequencing systems. Specifically, the statistical data quality value, the comparison rate, the GC content, the repetition rate, the genome coverage, the sequencing depth, and the like may be used according to the comparison result, and the sequencing data is quality-controlled according to the above information. At the same time, the useless data that cannot be compared or repeated is removed. Get a valid data set for subsequent analysis.

In addition, in an embodiment of the present invention, after obtaining the SNP information through the comparison, the obtained SNP information may also be filtered, for example, by pairing the sequencing results based on the preset filtering conditions. SNPs are known for filtration. According to an embodiment of the invention, the filtering conditions that can be employed are at least one of the following:

The SNP calling quality value is greater than 20. The term "SNP calling quality value" as used herein refers to the scoring result given to the confidence of each SNP during the operation of the SOAP analysis software.

SNP site sequencing depth is greater than 8;

The depth of the SNP locus is less than 5 times the average depth of the genome;

SNP site copy number is not greater than 2; and

The distance between the SNP site and the nearest other SNP site is greater than 5, and the expression "distance between two SNP sites" as used herein refers to the bases between the two SNP sites. The number, for example, "the distance is greater than 5", that is, the number of bases separated by two sites is greater than five.

Thereby, the obtained SNP results can be effectively filtered, so that the accuracy and efficiency of determining whether an individual has an abnormal state can be effectively improved.

S400: Determining an abnormal state related to a known SNP

As described above, after sequencing data is obtained by sequencing, and the sequencing data is analyzed, SNP information contained in the sequencing result can be obtained. Further, the SNP can be analyzed to determine whether the individual has an abnormal state associated with the SNP, such as whether or not there is a single-gene disease associated with the SNP.

According to an embodiment of the present invention, the type of SNP that can be employed is not particularly limited. In one embodiment of the invention, the known SNP is located in the human chromosome HBB gene region. In one embodiment of the present invention, the known SNP is at least one selected from the group consisting of: 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 located on human chromosome 11 at 5246716, 5246879, 5247026 Bit, 5248161 SNP. Thereby, it is possible to effectively determine whether the human body carries the SNP of the HBB gene region, thereby being able to effectively determine whether the subject has thalassemia, especially β-thalassemia.

Thus, the SNP information of the individual can be efficiently obtained by the above method, and the desired SNP information can be extracted for the relevant gene of the specific disease to be studied, and the typing result of the target gene can be obtained. After obtaining the typing result of the obtained target gene, the disease annotation can be performed by comparing with the existing database, and whether the corresponding SNP variation information causes the disease can be determined, and the embryo or individual corresponding to the single cell or the micro sample can be judged. For patients with Mendelian genetic disease (single gene-one disease), or the corresponding SNP mutation information does not cause disease, the corresponding embryo or individual is judged to be a carrier of the Mendelian genetic disease gene.

a system for determining whether an individual has an abnormal state

In another aspect of the invention, with reference to Figure 4, the present invention provides a system 1000 for determining whether an individual has an abnormal condition. According to an embodiment of the present invention, the system 1000 includes: a sequencing library construction device 100, a sequencing device 200,

The SNP determining device 300 and the abnormal state determining device 400.

According to an embodiment of the present invention, the sequencing library construction device 100 is configured to construct a sequencing library for a nucleic acid sample of an individual. According to an embodiment of the present invention, the sequencing device 200 is coupled to the sequencing library construction device 100, and thus, can be used to sequence the constructed sequencing library to obtain sequencing results composed of a plurality of sequencing data. In accordance with an embodiment of the present invention, SNP determining device 300 is coupled to the sequencing device for determining a known SNP contained in the sequencing result based on the obtained sequencing data. According to an embodiment of the present invention, the abnormal state determining device 400 is connected to the SNP determining device, thereby for determining whether the individual has the known SNP based on the previously determined known SNPs included in the sequencing result. Related exception status.

The method of determining whether an individual has an abnormal state described above can be effectively performed using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention, thereby efficiently determining the inclusion in the nucleic acid sample by sequencing.

SNPs, and since these SNPs are related to abnormal states, it is thereby possible to effectively determine whether the source individuals of the nucleic acid samples have abnormal states associated with these SNPs.

In an embodiment of the invention, the system may further comprise a nucleic acid sample extraction device 101. According to an embodiment of the invention, the nucleic acid sample extraction device 101 is adapted to extract at least a portion of the individual's whole genome DNA from a single cell or a micro sample of the individual. In one embodiment of the invention, the system 1000 can further comprise at least one single cell suitable for isolation from an individual selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres, and embryos. A biological sample separation device for micro-samples. Thus, the above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thus, the efficiency of determining whether an individual has an abnormal state can be improved by effectively predicting and evaluating whether an individual has an abnormal state by a small sample from an individual, and the cost of determining whether the individual has an abnormal state is reduced. In addition, these samples can be easily obtained from organisms, and can be specifically sampled for certain diseases to take specific analytical measures for certain specific diseases. In one embodiment of the invention, the biological sample separation device is adapted to separate at least one single cell selected from the group consisting of a dilution method, a mouth pipette separation method, a micromanipulation, a microdissection, a flow cytometry, and a microfluidic control. Or a small sample. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thereby, the single cells of the biological sample can be obtained efficiently and conveniently, so that the subsequent operations can be performed, whereby the single cells can be effectively separated from the individual, thereby further improving the efficiency of subsequently determining whether the individual has an abnormal state. In one embodiment of the present invention, the sequencing library construction device may further comprise a nucleic acid sample amplification unit adapted to amplify the nucleic acid sample to obtain a nucleic acid sample amplification product. In one embodiment of the invention, the amplification unit is adapted to perform at least one selected from the group consisting of PEP-PCR, DOP-PCR, OmniPlex WGA, and MDA. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state. The above-described method of determining whether an individual has an abnormal state can be effectively implemented using a system for determining whether an individual has an abnormal state according to an embodiment of the present invention. Thereby, the efficiency of amplifying whole genome DNA can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state. Herein, the operation mode of the "nucleic acid sample extraction device" is not particularly limited as long as the relevant nucleic acid sample can be obtained and the obtained nucleic acid sample is suitable for subsequent operations, for example, a whole genome from a single cell or a micro sample. DNA, can be released by lysing single cells with lysate and collecting single-cell whole genome DNA.

In addition, in an embodiment of the present invention, the sequencing library construction device may further include a screening unit. A nucleic acid probe is disposed in the screening unit to screen the nucleic acid sample amplification product with a nucleic acid probe to obtain a nucleic acid sample amplification product from a predetermined region; and to amplify a product for a nucleic acid sample from a predetermined region, The sequencing library was constructed. Thereby, the known SNPs included in the predetermined area can be effectively determined, whereby the efficiency of determining the abnormal state related to the SNPs according to the predetermined SNPs included in the predetermined area such as the exon area can be effectively improved. And accuracy. In one embodiment of the invention, the nucleic acid probe may be provided in the form of a chip. Thereby, the efficiency of screening by using a nucleic acid probe can be further improved, thereby further improving the efficiency of subsequently determining whether an individual has an abnormal state. In one embodiment of the present invention, the sequencing device is selected from Illumina Hiseq2000, Genome Analyzer. ^ At least one of a SOLiD sequencing system, an Ion Torrent, an Ion Proton, 454, a PacBio RS sequencing system, a Helicos tSMS sequencing device, and a nanopore sequencing device. Thereby, it is possible to utilize the high of these sequencing devices The characteristics of flux and deep sequencing further improve the efficiency of determining whether an individual has an abnormal state. In one embodiment of the invention, the sequencing is performed using an Illumina Hiseq 2000, the sequencing data being 90 bp in length. Thereby, the efficiency of subsequent SNP analysis can be further improved, thereby improving the efficiency of determining whether an individual has an abnormal state. In an embodiment of the present invention, the SNP determining apparatus further includes: a comparing unit configured to determine a known SNP included in the sequencing result by comparing the sequencing data with a reference gene . In one embodiment of the invention, the comparison unit is adapted to perform alignment using SOAP/SOAP2 software. Thereby, the sequencing data included in the sequencing result can be effectively analyzed, so that the SNP included in the sequencing result can be effectively determined, and the efficiency of determining whether the individual has an abnormal state can be improved.

In an embodiment of the present invention, the SNP determining apparatus may further include: a SNP filtering unit, wherein the SNP filtering unit is adapted to filter the known SNPs included in the sequencing result based on the following filtering conditions: SNP calling quality The value is greater than 20; the sequencing depth of the SNP site is greater than 8; the depth of the SNP site is less than 5 times the average depth of the genome; the copy number of the SNP site is not greater than 2; and the distance between the SNP site and the nearest other SNP site is greater than 5 . Thereby, the obtained SNP results can be effectively filtered, so that the accuracy and efficiency of determining whether an individual has an abnormal state can be effectively improved.

In one embodiment of the invention, the known SNP is located in the human chromosome HBB gene region. In one embodiment of the present invention, the known SNP is at least one selected from the group consisting of: 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, rs3397 1440, 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 SNPs located on human chromosome 11 at 5246716, 5246879, 5247026, and 5248161. Thus, the system for determining whether an individual has an abnormal state according to an embodiment of the present invention can effectively implement the foregoing A method of determining whether an individual has an abnormal state. Therefore, it can be effectively judged whether the subject has suffering from thalassemia, especially β-thalassemia.

The term "connected," as used herein, is to be understood broadly and can be either directly connected or indirectly connected, even using the same container or device, as long as functional linkages are possible, such as nucleic acid sample extraction and The nucleic acid sample amplification can be carried out in the same apparatus, that is, after the nucleic acid sample is extracted, the nucleic acid sample amplification processing can be performed in the same apparatus or container, and the extracted nucleic acid sample set does not need to be transported to other ones. Equipment or container, as long as the conditions within the device (including the reaction conditions and the composition of the reaction system) are converted to be suitable for nucleic acid sample amplification reaction, thus achieving the functional connection between nucleic acid sample extraction and nucleic acid sample amplification , can also be considered to be covered by the term "connected".

Those skilled in the art will appreciate that the features and advantages described above with respect to methods for determining whether an individual has an abnormal state, and of course the system of the present invention for determining whether an individual has an abnormal state, for convenience, are no longer Narration.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available, for example, from Illumina.

General method

As a non-limiting example, in a particular embodiment of the invention, single-cell disease is detected on a single cell or a microsample using a method comprising the following steps:

S1: single cell separation / micro sample preparation;

S2: whole genome amplification;

S3: sequencing sample preparation (library preparation);

S4: high throughput sequencing;

S5: Data comparison and quality control, obtaining a valid data set;

S6: SNP Calling;

S7: SNP extraction and filtration to obtain the typing result of the target gene;

S8: Disease note.

Example 1

Sample: human 5-8 cell stage blastomere single cell

Specific operational procedures: 1. Human 5-8 cell stage blastomere single cell separation

During the in vitro fertilization-embryo preimplantation genetic diagnosis (IVF-PGD) cycle, sperm and eggs are fertilized in vitro and cultured in vitro to the third day to form blastomeres at 5-8 cell stage. On the third day, a conventional biopsy was performed. Under the micromanipulator, a blastomere single cell was taken out, placed in a PCR tube containing the lysate, and stored at -80 °C. The blastomere after biopsy continues to culture until the fifth day, reaching the embryonic stage, can be vitrified, or used directly for implantation.

2. Whole genome amplification

Single-cell whole-genome amplification was performed using Qiagen's REPLI-g Mini Kit kit according to the manufacturer's protocol. The blastomere single cells were first subjected to alkaline lysis, and then the amplification reaction solution was added for constant temperature expansion at 30 °C. increase.

3. Sequencing sample preparation (library construction)

A DNA sequencing library was constructed using the Illumina Paired-End DNA Sample Prep Kit according to the manufacturer's protocol. Three libraries were prepared for the blastomere single-cell whole genome amplification products obtained in the previous section. The expected inserts of the library were 200 bp, 350 bp, and 500 bp, respectively. The actual insert size is shown in Table 1.

4. High-throughput sequencing

High throughput sequencing was performed using the Illumina Hiseq 2000 sequencing system. In this example, a strategy for whole genome sequencing of blastomere single cells is employed. The well-prepared library of blastomere single-cell amplification products was prepared by cBot, and then run on the Hiseq2000 sequencer. The sequencing length was 90 bp, and the Pair End was sequenced in two directions. One lane was measured for each library, and three lanes were measured.

5. Data comparison and quality control, obtaining a valid data set

Sequencing data were aligned using SOAP2 software and referenced to the human reference genome sequence (Hgl9; NCBI Build37), allowing up to two base mismatches when aligned. The raw data quality, GC content, actual insert fragment size, alignment rate, repetition rate, and genomic coverage and sequencing depth were calculated based on the alignment results. See Table 1 for specific information. The quality of the sequencing data is controlled by these statistical results. In this example, we obtained a total of 38.25X of the whole genome, and the statistical results of the data were all up to standard. After that, the data of the upper reference genome and the repeated data are not removed, and a valid data set is obtained for SNP analysis.

Table 1 Comparative results of blastomere single cell sequencing data in Example 1 *

Sample PE 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)

Marking:

* BLSl is the number of single-cell samples of blastomeres, BLSl(total) is the statistical result of the combination of three lane data of BLSl sample, and Q20 (%) is the ratio of the data of quality value above 20 to the total amount of data. GC (%) indicates the actual GC content percentage of the sequencing data, Insert Size indicates the actual library insert size of the sequencing data, Clean Reads indicates the amount of read data remaining after the low-quality read is removed, and PE-alignment indicates both ends of the Pair End. Both can read the ratio of the read data of the reference genome to the total data volume, PE reads that both ends of the Pair End can compare the read data of the reference genome, and PE Unique means that both ends of the Pair End can be uniquely aligned to the reference genome. The amount of read data, Coverage represents the coverage of the whole genome, Mean Depth represents the average depth of the whole genome, and Duplication Rate represents the ratio of repeated read data to total read data.

6. SNP Calling

This embodiment uses the SOAPsnp software to perform SNP Calling on the valid data sets obtained above according to the instructions of SOAPsnp (see http://soa.genomics.org.cn/soapsn, html, which is incorporated herein by reference). Finally, the SNP data set is obtained.

7. SNP extraction and filtration to obtain the genotyping results of the target gene

The SNP data set obtained above is filtered, and the filtering conditions are as follows:

a. SNP calling quality value is greater than 20;

b. The sequencing depth of the site is greater than 8;

c. The depth of the site is less than 5 times the average depth of the genome;

d. The copy number of the site is not more than 2;

e. The distance between the SNP and the nearest SNP is greater than 5.

The filtered SNP data extracts SNPs located in the target gene region for the disease-related genes to be detected. In the present example, the gene for β-thalassemia is detected to detect whether the corresponding blastomere embryo has β-sea aquaemia, or whether it is a carrier of the β thalassemia disease gene. Therefore, in this example, the SNP locus located in the ΗΒΒ gene region of chromosome 11 was extracted, and the specific information is shown in Table 2.

Table 2 SNP site information located in the ΗΒΒ gene region obtained in Example 1 *

/ZT0ZN3/X3d £606 Please ΪΟΖ OAV

/ZT0ZN3/X3d £606 Please ΪΟΖ OAV 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 :

^Chromosome indicates the chromosome number, Locus indicates the site number of the base corresponding to the SNP on the chromosome, Ref indicates the base type of the corresponding site on the human reference genome in the database, and Blastomere indicates the corresponding SNP position in the blastomere single cell data. The type information of the point, Mutation indicates the mutation type of the corresponding site existing in the database, the SNP ID indicates the ID number of the SNP site in the database, and Gene indicates which gene region the SNP site is located in.

8. Disease notes

The disease annotation is further made based on the SNP information of the target gene filtered and extracted as described above. In this example, heterozygous SNP variants were found in the 5247141 and 5247791 bases of chromosome 11, respectively, which are located in the HBB gene region. Homozygous mutations at these two sites can cause beta thalassemia. In the present example, the two sites are heterozygous mutations, indicating that the blastomeres corresponding to the single cells are carriers of the β-thalassemia pathogenic gene.

Example 2

Sample: normal human blood single cell

Specific operational procedures:

1. Normal human blood single cell separation

The blood sample of this example is derived from a human individual with a normal phenotype. A small amount of blood sample was taken and centrifuged to separate the leukocyte layer. The leukocytes were washed with PBS, suspended in PBS droplets, and the individual leukocytes were separated by a mouth pipe, placed in 1-2 μl of alkaline cell lysate, and frozen at -80 ° C for more than 30 min.

2. Whole genome amplification

Qiagen's REPLI-g Mini Kit kit was used. According to the manufacturer's protocol, blood single cells were subjected to alkaline lysis after treatment at 65 °C for 10 minutes, and then amplified reaction solution was added for constant temperature amplification at 30 °C.

3. Chip capture library construction

Whole-genome amplification products of blood single-cell samples are chip-captured to specific target regions, and library construction is performed simultaneously. The Agilent chip used in this example targets a target area of 2.1 M in size, including all exon regions of one hundred single gene disease-associated genes. For the method of using the Agilent chip, refer to the manufacturer's instructions. The captured and constructed sequencing libraries were subjected to high throughput sequencing.

4. High-throughput sequencing

This example uses the Illumina Hiseq 2000 sequencing system for high throughput sequencing. In this embodiment, a strategy for chip capture sequencing of blood single cells is adopted, and the target region is all exon regions of one hundred single gene disease-related genes, about 2.1 M in size. A well-prepared chip capture library prepared by blood single-cell amplification products was prepared by cBot, and then run on a Hiseq2000 sequencer. The sequencing length was 90 bp, and Pair End was bidirectionally sequenced. The amount of sequencing data was expected to be 1 to 2 G bases.

5. Data comparison and quality control, obtaining a valid data set

Sequencing data were aligned using SOAP2 software and referenced to the human reference genome sequence (Hgl9; NCBI Build37), allowing up to two base mismatches when aligned. The raw data quality, GC content, alignment rate, repetition rate, and genomic coverage and sequencing depth were calculated based on the alignment results. The specific information is shown in Table 3. The quality of the measured data is controlled by these statistical results. In this embodiment, we obtain a total amount of data of 457X in the target area, and the statistical results of the data can reach the standard. After that, the data of the upper reference genome and the repeated data are not removed, and a valid data set is obtained for SNP analysis.

Table 3 Comparative results of blood single cell sequencing data in Example 2

Marking:

*GC (%) indicates the actual GC content percentage of the sequencing data, Reads indicates the amount of read data remaining after the read of the low quality value is removed, Production indicates the amount of base data calculated according to the Reads value, and PE-alignment indicates the both ends of the Pair End Both can compare the ratio of the read data of the reference genome to the total data volume, PE Unique indicates that the pair of Pair ends can uniquely compare the read data to the reference genome, Coverage indicates the coverage of the whole genome, and Mean Depth indicates the whole genome. The average depth, Duplication Rate indicates the ratio of the repeated read data to the total read data, and the specificity (Reads) indicates the ratio of the read data amount of the target area to the total read data. The specificity (Bases) indicates the specific alignment. The ratio of the amount of base data in the target area to the total amount of base data.

6. SNP Calling

This embodiment uses the SOAPsnp software according to the instructions of SOAPsnp (see http://soap.genoniics, org m''soapsnp.htrnl, which is incorporated herein by reference) for the valid data set obtained above. Perform SNP Calling and finally obtain the SNP data set.

a. SNP calling quality value is greater than 20;

b. The sequencing depth of the site is greater than 8;

c. The depth of the site is less than 5 times the average depth of the target region;

d. The copy number of the site is not more than 2;

e. The distance between the SNP and the nearest SNP is greater than 5.

The filtered SNP data extracts SNPs located in the target gene region for the disease-related genes to be detected. In the present example, the gene for β-thalassemia is detected to detect whether the corresponding blastomere embryo has β-sea aquaemia, or whether it is a carrier of the β thalassemia disease gene. Therefore, in this example, the SNP locus located in the ΗΒΒ gene region of chromosome 11 was extracted, and the specific information is shown in Table 4.

Table 4 SNP site information located in the ΗΒΒ gene region in Example 2

0/ZT0ZN3/X3d £606 Please ΪΟΖ OAV 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 chr 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ->G rs34305195 HBB

* Chromosome indicates the chromosome number, Locus indicates the site number of the base corresponding to the SNP on the chromosome, Ref indicates the base type of the corresponding site on the human reference genome in the database, and Blood Cell indicates the corresponding SNP site in the blood single cell data. The type information, Mutation indicates the type of mutation of the corresponding site existing in the database, the SNP ID indicates the ID number of the SNP site in the database, and Gene indicates which gene region the SNP site is located in.

8. Disease notes

The SNP information of the target gene filtered and extracted above is further subjected to disease annotation. In this example, no heterozygous or homozygous mutation site was detected in the HBB gene region, indicating that the individual corresponding to the blood single cell in the present example is not a beta thalassemia disease patient, nor is it a beta thalassemia disease gene carrier. The beta thalassemia disease gene region is a normal genotype. To this end, embodiments of the present invention enable a method for detecting Mendelian genetic diseases (single-gene diseases) on single-cell or micro-samples based on high-throughput sequencing.

Industrial applicability

The invention can be effectively applied to the construction and sequencing of a DNA sequencing library of sample DNA, and the obtained library has good quality and accurate sequencing results.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

claims

1. A method for determining whether an individual suffers from an abnormal state, characterized by including:

Construct a sequencing library for the nucleic acid sample of the individual;

Sequencing the sequencing library to obtain sequencing results, where the sequencing results are composed of multiple sequencing data; determining known SNPs included in the sequencing results based on the sequencing data; and

Based on the known SNP, it is determined whether the individual suffers from an abnormal condition associated with the known SNP.

2. The method according to claim 1, characterized in that the individual is a human.

3. The method according to claim 1, characterized in that the abnormal state is a disease.

4. The method according to claim 3, characterized in that the disease is a single gene disease.

5. The method according to claim 4, characterized in that the single gene disease is at least one selected from the group consisting of thalassemia and erythrocyte glucose-6-phosphate dehydrogenase deficiency.

6. The method according to claim 5, wherein the thalassemia is β-thalassemia.

7. The method according to claim 1, characterized in that the nucleic acid sample is at least a part of the individual's whole genome DNA.

8. The method according to claim 1, characterized in that the nucleic acid sample is extracted from an individual's single cell or a trace sample.

9. The method according to claim 8, characterized in that the single cell or trace sample is isolated from at least one selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres and embryos of the individual of.

10. The method according to claim 9, characterized in that the single cells are separated by at least one method selected from the group consisting of dilution method, mouth pipette separation method, visual manipulation, visual cutting, flow cytometry, and microfluidics. Detached.

11. The method according to claim 1, wherein constructing a sequencing library for the nucleic acid sample of the individual further includes:

Amplify the nucleic acid sample to obtain a nucleic acid sample amplification product; and

The sequencing library is constructed based on the nucleic acid sample amplification product.

12. The method according to claim 11, wherein the nucleic acid sample is whole genome DNA extracted from an individual's single cell,

Its scarf,

Amplification of the whole genome DNA is performed by at least one selected from the group consisting of PEP-PCR, DOP-PCR, OmniPlex WGA and MDA.

13. The method according to claim 11, characterized in that, before constructing the sequencing library for the nucleic acid sample amplification product, further comprising: Use nucleic acid probes to screen the nucleic acid sample amplification products to obtain nucleic acid sample amplification products from a predetermined region; and

The sequencing library is constructed based on the nucleic acid sample amplification product from the predetermined region.

14. The method according to claim 13, characterized in that the nucleic acid probe is provided in the form of a chip.

15. The method according to claim 13, characterized in that the predetermined region is at least one exon region.

16. The method according to claim 1, characterized in that the sequencing is performed using a sequencing system selected from the group consisting of Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing system, Ion Torrent, Ion Proton, 454, PacBio RS sequencing system, Helicos tSMS technology and Nano performed by at least one of the well sequencing technologies.

17. The method according to claim 16, wherein the sequencing is performed using Illumina Hiseq2000, and the length of the sequencing data is 90 bp.

18. The method of claim 1, wherein determining the known SNPs contained in the sequencing results based on the sequencing data is performed by comparing the sequencing data with a reference gene. of.

19. The method of claim 18, wherein the reference gene is a known human genome sequence.

20. The method according to claim 18, characterized in that the comparison is performed by SOAP/SOAP2 software.

21. The method according to claim 20, further comprising filtering known SNPs contained in the sequencing results, and the filtering is based on the following filtering conditions:

SNP calling quality value is greater than 20;

SNP site sequencing depth is greater than 8;

The SNP site depth is less than 5 times the average depth of the genome;

The copy number of the SNP locus is not greater than 2; and

The distance between the SNP site and the nearest other SNP site is greater than 5.

22. The method according to claim 1, characterized in that the known SNP is located in the HBB gene region of the human chromosome.

23. The method according to claim 22, characterized in that the known SNP is at least one selected from the following: rs33985472, rs63750954, rs63751128, rs33978907, rs34029390, rs34809925, rs33953406, rs33910569, rs33910 569, rs33971634, rs3392539 rs33946267, rs35485099, rs36015961, rs33930977, rs35256489, rs33952266, rs33952266, rs33952266, rs33914668, rs33914668, rs33913413, rs3 107686 83. 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, rs3 3916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs339 41849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628, rs34305195 and located on human chromosome 11 at positions 5246716, 5246879 and 5247026 , 5248161 SNPs.

24. A system for determining whether an individual suffers from an abnormal state, characterized by: including:

A sequencing library construction device, the sequencing library construction device is used to construct a sequencing library for the nucleic acid sample of the individual;

A sequencing device, the sequencing device is connected to the sequencing library construction device, and is used to sequence the sequencing library to obtain a sequencing result, where the sequencing result is composed of multiple sequencing data;

SNP determination device, the SNP determination device is connected to the sequencing device, and is used to determine the known SNP included in the sequencing result based on the sequencing data; and

Abnormal state determining device, the abnormal device determining device is connected to the SNP determining device, and is used to determine whether the individual suffers from an abnormal state related to the known SNP based on the known SNP.

25. The system according to claim 24, further comprising:

Nucleic acid sample extraction device, the nucleic acid sample extraction device is suitable for extracting at least a part of the individual's whole genome DNA from the individual's single cells or trace samples.

26. The system according to claim 25, further comprising:

A biological sample separation device, the biological sample separation device is adapted to separate single cells or Z amount of samples from at least one selected from the group consisting of blood, tissue, urine, gametes, fertilized eggs, blastomeres and embryos of an individual.

27. The system according to claim 26, characterized in that the biological sample separation device is suitable for at least one selected from the group consisting of dilution method, mouth pipette separation method, visualization operation, visualization cutting, flow cytometry, and flow control. A method of isolating single cells or trace samples.

28. The system according to claim 24, wherein the sequencing library construction device further includes: a nucleic acid sample amplification unit, the nucleic acid sample amplification unit is adapted to amplify the nucleic acid sample to obtain Nucleic acid sample amplification products.

29. The system of claim 24, wherein the amplification unit is adapted to perform a process selected from the group consisting of PEP-PCR,

At least one of DOP-PCR, OmniPlex WGA and MDA.

30. The system according to claim 29, characterized in that the sequencing library construction device further includes: a screening unit, and a nucleic acid probe is provided in the sorting unit to use the nucleic acid probe to detect the nucleic acid. Screening the sample amplification products to obtain nucleic acid sample amplification products from a predetermined region; and

31. The system according to claim 30, characterized in that the nucleic acid probe is provided in the form of a chip.

32. The system according to claim 24, wherein the sequencing device is selected from the group consisting of Illumina Hiseq2000, Genome Analyzer, SOLiD sequencing system, Ion Torrent, Ion Proton, 454, PacBio RS sequencing system, Helicos tSMS sequencing device, and At least one nanopore sequencing device.

33. The system according to claim 24, characterized in that the SNP determination device further includes: a comparison unit, the comparison unit is used to determine the sequencing data by comparing the sequencing data with a reference gene. Known SNPs included in the results.

34. The system according to claim 33, characterized in that the comparison unit is adapted to use SOAP/SOAP2 software for comparison.

35. The system according to claim 33, characterized in that the SNP determination device further includes: a SNP filtering unit, the SNP filtering unit is adapted to filter known genes contained in the sequencing results based on the following filtering conditions. SNP filtering:

SNP calling quality value is greater than 20;

SNP site sequencing depth is greater than 8;

The SNP site depth is less than 5 times the average depth of the genome;

The copy number of the SNP locus is not greater than 2; and

36. The system according to claim 24, characterized in that the known SNP is located in the HBB gene region of the human chromosome.

37. The system according to claim 36, characterized in that the known SNP is at least one selected from the following: rs33985472, rs63750954, rs63751128, rs33978907, rs34029390, rs34809925, rs33953406, rs33910569, rs33910 569, rs33971634, rs3392539 rs33946267, rs35485099, rs36015961, rs33930977, rs35256489, rs33952266, rs33952266, rs33952266, rs33914668, rs33914668, rs33913413, rs3 3913413, rs34483965, rs34793594, rs35703285, rs63750433, rs34690599, rs63751175, rs35328027, rsl609812, rs34451549, rs7480526, rsl076868 3. rs35099082, rs63750283, rs63750283, rs33945777, rs33945777, rs33945777, rs33933298, rs33913712, rs33995148, rs33931779, rs33969400, rs33922842, rsll549407, rs33 974936, 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, rs3 3916412, rs35424040, rs33950507, rs33950507, rs33951465, rs33959855, rs33972047, rs33986703, rs3471601 rs63750783, rs35799536, rs33930702, rs33930702, rs33930702, rs339 41849, rs33941849, rs33941849, rs34563000, rs34135787, rs34704828, rs63750628, rs34305195 and located on human chromosome 11 at positions 5246716, 5246879 and 5247026 , 5248161 SNPs.