CN108363903A - One kind being suitable for single celled chromosomal aneuploidy detecting system and application - Google Patents
One kind being suitable for single celled chromosomal aneuploidy detecting system and application Download PDFInfo
- Publication number
- CN108363903A CN108363903A CN201810078283.XA CN201810078283A CN108363903A CN 108363903 A CN108363903 A CN 108363903A CN 201810078283 A CN201810078283 A CN 201810078283A CN 108363903 A CN108363903 A CN 108363903A
- Authority
- CN
- China
- Prior art keywords
- window
- section
- data amount
- relative data
- chromosomal aneuploidy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000036878 aneuploidy Diseases 0.000 title claims abstract description 75
- 231100001075 aneuploidy Toxicity 0.000 title claims abstract description 71
- 230000002759 chromosomal effect Effects 0.000 title claims abstract description 58
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 210000000349 chromosome Anatomy 0.000 claims abstract description 42
- 238000012163 sequencing technique Methods 0.000 claims abstract description 31
- 238000005192 partition Methods 0.000 claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims description 69
- 238000000034 method Methods 0.000 claims description 50
- 238000004519 manufacturing process Methods 0.000 claims description 42
- 238000012937 correction Methods 0.000 claims description 37
- 238000002156 mixing Methods 0.000 claims description 37
- 238000003908 quality control method Methods 0.000 claims description 33
- 238000013412 genome amplification Methods 0.000 claims description 30
- 230000015556 catabolic process Effects 0.000 claims description 23
- 238000010276 construction Methods 0.000 claims description 16
- 230000002159 abnormal effect Effects 0.000 claims description 14
- 108700039887 Essential Genes Proteins 0.000 claims description 10
- 238000001787 Wald–Wolfowitz test Methods 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 7
- 238000004364 calculation method Methods 0.000 claims description 6
- KJDGFQJCHFJTRH-YONAWACDSA-N 16-Ketoestradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](C(=O)C4)O)[C@@H]4[C@@H]3CCC2=C1 KJDGFQJCHFJTRH-YONAWACDSA-N 0.000 claims description 5
- 238000012268 genome sequencing Methods 0.000 claims 2
- 238000005516 engineering process Methods 0.000 abstract description 10
- 230000007812 deficiency Effects 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 89
- 239000000047 product Substances 0.000 description 71
- 108020004414 DNA Proteins 0.000 description 23
- 239000000203 mixture Substances 0.000 description 23
- 210000002308 embryonic cell Anatomy 0.000 description 14
- 239000011324 bead Substances 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 230000003321 amplification Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 210000001161 mammalian embryo Anatomy 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 108010077544 Chromatin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000003483 chromatin Anatomy 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000011017 operating method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000003322 aneuploid effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 102100030374 Actin, cytoplasmic 2 Human genes 0.000 description 2
- 108010032769 Autophagy-Related Protein 8 Family Proteins 0.000 description 2
- 102000007353 Autophagy-Related Protein 8 Family Human genes 0.000 description 2
- 102100039924 Cytochrome b-c1 complex subunit 1, mitochondrial Human genes 0.000 description 2
- 102100031655 Cytochrome b5 Human genes 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- 101000773237 Homo sapiens Actin, cytoplasmic 2 Proteins 0.000 description 2
- 101000607486 Homo sapiens Cytochrome b-c1 complex subunit 1, mitochondrial Proteins 0.000 description 2
- 101000922386 Homo sapiens Cytochrome b5 Proteins 0.000 description 2
- 101000573220 Homo sapiens NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 Proteins 0.000 description 2
- 101000987578 Homo sapiens Peripherin Proteins 0.000 description 2
- 208000017924 Klinefelter Syndrome Diseases 0.000 description 2
- 108091036060 Linker DNA Proteins 0.000 description 2
- 102100026374 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 Human genes 0.000 description 2
- 102100028465 Peripherin Human genes 0.000 description 2
- 208000037280 Trisomy Diseases 0.000 description 2
- 208000026928 Turner syndrome Diseases 0.000 description 2
- 210000001766 X chromosome Anatomy 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 210000000982 limb bud Anatomy 0.000 description 2
- 238000001531 micro-dissection Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- ODLMAHJVESYWTB-UHFFFAOYSA-N propylbenzene Chemical compound CCCC1=CC=CC=C1 ODLMAHJVESYWTB-UHFFFAOYSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 201000010374 Down Syndrome Diseases 0.000 description 1
- 101001030211 Homo sapiens Myc proto-oncogene protein Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000034655 MIF Human genes 0.000 description 1
- 108060004872 MIF Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 101710179516 Run domain Beclin-1-interacting and cysteine-rich domain-containing protein Proteins 0.000 description 1
- 102100030852 Run domain Beclin-1-interacting and cysteine-rich domain-containing protein Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000003698 laser cutting Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007838 multiplex ligation-dependent probe amplification Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 210000003765 sex chromosome Anatomy 0.000 description 1
- 230000036301 sexual development Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 206010044689 trisomy 22 Diseases 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Medical Informatics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Theoretical Computer Science (AREA)
- Evolutionary Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Artificial Intelligence (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Data Mining & Analysis (AREA)
- Databases & Information Systems (AREA)
- General Engineering & Computer Science (AREA)
- Evolutionary Computation (AREA)
- Bioethics (AREA)
- Public Health (AREA)
- Software Systems (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to gene engineering technology fields, and in particular to one kind being suitable for single celled chromosomal aneuploidy detecting system and application.The detecting system includes window division module, section partition module, section copy rate computing module, section aneuploidy determination module.The detecting system uses most 700k reads/ samples, you can chromosome deficiency/repetition to the pattern detection 5Mb or more, accuracy rate reduce sequencing depth, reduce cost up to 99.9%.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind being suitable for single celled chromosomal aneuploidy and examines
Examining system and application.
Background technology
Aneuploid refers to lacking or additionally increasing by one or several chromosomes in euploid chromosomal.Normally due to
Pair of homologous ploidy level or detach and formed the gamete of numerical abnormalities of chromosomes in advance when meiosis, this kind of gamete that
This combines or is combined with normal gamete, generates various aneuploid cells.In addition it also will produce non-multiple in somatic cell division
Body cell, such as the very high tumour cell of aberration rate.
Some genetic diseases are closely related with the mankind for non-multiple chromosome.Most common such as Down syndrome, incidence is about
1/800, caused by more No. 21 chromosomes and 13 3 bodies and 18 Trisomies, respectively because having more one No. 13
Occur miscarrying with No. 18 chromosomes.Autosome heteroploidy is also the broad aspect reason for causing Pregnancy failure and miscarrying.
Sex chromosome numerical abnormality can cause sexual development abnormelity.The individual of the more X chromosomes (47, XXY) of male is congenital testis
Ball hypoplasia disease (Klinefelter syndromes).Turner syndromes are also known as Turner syndrome syndrome, due to
Lack an X chromosome, caryogram 45, X.
Embryo be for referring exclusively to zoogamy refer to male sex-cell and female sex cell be combined into zygote it
Afterwards, what is formed after multiple cell division and cell differentiation has the young body for the ability for developing into biological adult.Embryo refers to just
It is the initial period of generative propagation development forming process, the first division since fertilized eggs develops to next stage before starting,
It is the Developmental Biology earliest stage.
Cell is the base unit of life composition, and a full set of genome at base unit.At present before Embryonic limb bud cell
Science of heredity detection is required for carrying out in single (or multiple) cellular level.In individual cell level analysis genome at detection dye
Whether colour solid is normal, is also common research method.
The method of traditional detection embryo's aneuploid include fluorescence in situ hybridization (FISH), realtime-PCR, MLPA,
Biochip etc..Biochip is divided into comparative genomic hybridization hybrid chip and SNP chip, has become the primary hand of heteroploid detection
Section, but its flux is low, can only once detect limited embryo, of high cost, operation is relative complex.FISH and realtime-PCR
As faster molecular biology for detection, be applied to more than 80% heteroploid detection in, but they by
The limitation of method number of probes itself all can not achieve while carry out complete detection to all 23 pairs of chromosomes, and flux is very low.
New-generation sequencing technology is fast-developing, and the application in chromosome detection is also more and more.Dennis Lo et al.
Develop the method being detected to free nucleic acid in Maternal plasma based on Illumina GA high-flux sequences, detects No. 21 dyes
The accuracy of three body of colour solid is up to 99% or more.Aneuploid detection for embryo, although also occurring being based partially on new one
For the detection method of sequencing technologies, but it is all perfect not to the utmost from detection speed, operation complexity, cost etc..
Invention content
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide one kind being suitable for single celled dyeing
Body aneuploidy detecting system and application, quickly to detect the isocellular chromosomal aneuploidy of embryonic cell, reproduction cell.
In order to achieve the above objects and other related objects, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides one kind and being suitable for single celled chromosomal aneuploidy detecting system, including：
Window division module, for the partition window in the sequence of reference gene group；Section partition module, for according to sample to be detected
The relative data amount ratio that each window obtained is calculated with normal sample determines breakpoint location, and according to breakpoint location dividing regions
Section；Wherein, the normal sample does not have chromosomal aneuploidy；Section copy rate computing module is copied for calculation of sector
Rate CRj；Section aneuploidy determination module, for judging whether section has chromosomal aneuploidy according to section copy rate.
In one possible implementation, in the section partition module, the relative data amount ratio is using following
Formula calculates：
ri=Wi/N
WiExpression falls into the ordered sequence number of i-th of window, and 1≤i≤S, S are window sum；
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence is surveyed for full-length genome
The sequence to the reference gene group is compared in the sequence that sequence obtains；
riIndicate the relative data amount of i-th of window；
For the relative data amount of i-th of window of sample to be detected；
For the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
In one possible implementation, the relative data amount ratio ViG/C content correction is carried out, each window is obtained
Correction relative data amount ratio Vi', correction relative data amount ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeTo fall into all window V in e-th of G/C content sectioniThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sample to be detectediThe mean value of value,
Vi' be i-th of window correction relative data amount ratio.
In one possible implementation, in the section partition module, the breakpoint location refers to relative data amount
Ratio has the boundary of two adjacent window apertures of significant difference.
In one possible implementation, in the section partition module, the breakpoint location is true by following method
It is fixed：Using n adjacent window as window group, S window is divided into m window group, m=S-n+1, m are window group
Body sum；S is the window sum that sample divides；N is window number contained in a window group, then utilizes each window group
In each window relative data amount ratio ViNonparametric runs test is carried out, the P values of each adjacent window apertures group are calculated, if P values are small
In threshold value, then judge that the intersection of corresponding adjacent window apertures group is the breakpoint location of copy number variation (CNV)；Wherein, P values are aobvious
Write sex differernce P values.
In one possible implementation, the section copy rate refers to each window relative data amount ratio in section
Average value.
In one possible implementation, the section copy rate is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, by calculating being averaged for each window relative data amount ratio in j-th of section
Value obtains.
In one possible implementation, in the section aneuploidy determination module, if 0.75<CRj<1.25 then
Judge that j-th of section is normal；If CRj≤ 0.75 or CRj>=1.25, then judge j-th of section for non-integral multiple exception.
In one possible implementation, if j-th of section corresponds to whole chromosome, it can determine that the whole chromosome
Whether it is that non-integral multiple is abnormal.
In one possible implementation, the detecting system further includes：Cell separation module, cell full-length genome expand
Increase module, cell amplified production quality Control module, library construction module, high-flux sequence module.
In one possible implementation, the cell amplified production quality Control module is used for the house-keeping gene using cell
Quality Control is carried out to the whole genome amplification product of cell, and judges whether Quality Control result meets preset condition, if met default
Condition then illustrates that the whole genome amplification product is qualified amplified production.
In one possible implementation, the library construction module is used to build the sequencing of the qualified amplified production
Library, the library construction module include：Broken submodule, connector connection submodule, mixing submodule, mix products purifying
Module, PCR enrichment submodules and enriched product purify submodule, and broken submodule is obtained for being crushed the qualified amplified production
Obtain breakdown products；The connector connection submodule is used to add connector for the breakdown products, obtains connector breakdown products；It is described
The connector breakdown products that submodule is used for the multiple samples of mixed in equal amounts are mixed, mix products are obtained；The mix products are pure
Beggar's module is for purifying the mix products；The PCR enrichment submodules are pure for being enriched with the mix products purification blocks
Mix products after change, obtain enriched product；The enriched product purifying submodule obtains institute for purifying the enriched product
State sequencing library.
Second aspect of the present invention provides one kind and being suitable for single celled chromosomal aneuploidy detection method, including as follows
Step：S1, the partition window in the sequence of reference gene group obtain S window；S2, according to sample to be detected and normal sample
The relative data amount ratio for calculating each window obtained determines breakpoint location, and divides section according to breakpoint location；Wherein, described
Normal sample does not have chromosomal aneuploidy；Wherein, the normal sample does not have chromosomal aneuploidy；S3, area is calculated
Section copy rate CRj；S4, the whether specific chromosomal aneuploidy of section is judged according to section copy rate.
In one possible implementation, the relative data amount ratio is calculated using following equation：
ri=Wi/N
WiIndicate to fall into the ordered sequence number of i-th of window, 1≤i≤S,
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence is surveyed for full-length genome
The sequence to the reference gene group is compared in the sequence that sequence obtains；
riIndicate the relative data amount of i-th of window；
For the relative data amount of i-th of window of the sample to be detected；
For the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
In one possible implementation, the relative data amount ratio ViG/C content correction is carried out, each window is obtained
Correction relative data amount ratio Vi', correction relative data amount ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeTo fall into all window V in e-th of G/C content sectioniThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sampleiThe mean value of value,
Vi' be i-th of window correction relative data amount ratio.
In one possible implementation, the breakpoint location, which refers to relative data amount ratio, has significant difference
The boundary of two adjacent window apertures.
In one possible implementation, the breakpoint location is determined by following method：It will be adjacent in S window
N window is divided into a window group, obtains m window group, m=S-n+1；M is window group sum；S is the window
The window sum that mouth division module divides；N is window number contained in a window group；Then it utilizes each in each window group
The relative data amount ratio V of windowiNonparametric runs test is carried out, the P values of each adjacent window apertures group are calculated, if P values are less than threshold
Value then judges that the intersection of corresponding adjacent window apertures group is the breakpoint location of copy number variation (CNV)；Wherein, P values are conspicuousness
Difference P values.
In one possible implementation, the section copy rate refers to each window relative data amount ratio in section
Average value.
In one possible implementation, the section copy rate CRjIt is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, CRjBy calculating the flat of each window relative data amount ratio in j-th of section
Mean value obtains.
In one possible implementation, in step S4, if 0.75<CRj<1.25, then judge that j-th of section is just
Often；If CRj≤ 0.75 or CRj>=1.25, then judge j-th of section for non-integral multiple exception.
In one possible implementation, if j-th of section corresponds to whole chromosome, it can determine that the whole chromosome
Whether it is that non-integral multiple is abnormal.
In one possible implementation, described to further include suitable for single celled chromosomal aneuploidy detection method
Following steps：Cell detaches；Cell whole genome amplification；Cell amplified production Quality Control；Library construction；High-flux sequence.
In one possible implementation, the cell amplified production Quality Control, for the house-keeping gene pair using cell
The whole genome amplification product of cell carries out Quality Control, and judges whether Quality Control result meets preset condition, and item is preset if met
Part then illustrates that the whole genome amplification product is qualified amplified production.
In one possible implementation, the library construction, including DNA is broken, connector connection, mixing, mixes production
Object purifying, PCR enrichments and enriched product purifying, the DNA broken refers to the broken qualified amplified production, obtains broken production
Object；The connector connection refers to adding connector for the breakdown products, obtains connector breakdown products；The mixing refers to that equivalent is mixed
The connector breakdown products for closing multiple samples, obtain mix products；The mix products purifying refers to the purifying mixing production
Object；The enriched product purifying refers to the purifying enriched product, obtains sequencing library.
Compared with prior art, provided by the invention to be suitable for single celled chromosomal aneuploidy detecting system and side
Method has the advantages that：
(1), required sample initial amount is low, and the chromosomal aneuploidy of detection individual cells may be implemented.
(2), window size can be set according to demand, and the size by controlling window improves detection resolution.
(3), whole genome amplification product Quality Control
After embryonic cell completes single cell whole genome amplification, Quality Control will be carried out to amplified production, to evaluate full genome
Whether group amplification procedure succeeds, such as unsuccessful, then terminates experiment, reduce cost consumption.
(4), it is enriched with after first sample mixing
During building library, mixed in equal amounts first is carried out to all sample DNAs after jointing, then carries out PCR enrichments,
The data volume of each sample output can be made more uniform using this method, avoid unnecessary data from wasting, and can subtract
Cost consumption after few enrichment experiment, reduces operating procedure, reduces operation complexity.
(5), 1ng originates DNA and builds library
Cell sample belongs to trace sample, and amount of DNA is few, and detection can be completed in entire detection process, 1ng DNA, reduces
The waste of sample.
(6), the reads numbers of 700k can complete the variation detection of 5Mb or more
Use most 700k reads/ samples, you can chromosome deficiency/repetition to the pattern detection 5Mb or more, it is accurate
True rate is up to 99.9%.Sequencing depth is reduced, cost is reduced.
(7), sample data is corrected
Because cell sample passes through whole genome amplification, many amplification deviations can be introduced, this method introduces sample data correction,
Reduce influence of these deviations to result.
Description of the drawings
Fig. 1 is contaminated suitable for unicellular chromosomal aneuploidy detecting system before detecting Embryonic limb bud cell to be using the present invention
The implementation process schematic diagram of colour solid aneuploidy.
Fig. 2：The amplification principle schematic of MDA.
Fig. 3：The distribution schematic diagram of relative data amount ratio before G/C content correction.
Fig. 4：The distribution schematic diagram of correction relative data amount ratio after G/C content correction.
Fig. 5：The chromosome testing result figure of T22 exception embryonic cells.
Fig. 6：The chromosome testing result figure of fragment deletion/repetition embryonic cell.
Fig. 7：The chromosome testing result figure of normal embryonic cells.
Specific implementation mode
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text
In addition explicitly point out, singulative "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,
Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
An embodiment of the present invention provides one kind being suitable for single celled chromosomal aneuploidy detecting system, including：Window
Division module, section partition module, section copy rate computing module, section aneuploidy determination module.
Window division module is used for the partition window in the sequence of reference gene group；Window is divided there are many optional mode,
It in one embodiment, can be according to the equal partition window of base number of reference gene group in each window；Implement in another kind
In mode, the equal model split window of the ordered sequence number of each window can be fallen into the sequence of normal sample；In another kind
In embodiment, the equal model split window of the ordered sequence number of each window can be fallen into the simulated series of reference gene group
Mouthful；Base number or the ordered sequence number fallen in single window can be set as required in single window, such as can take 50-
Any number in 1000kb, specifically, for example choosing 500kb, digital smaller under normal circumstances, as a result resolution ratio is higher；Its
In, normal sample is without chromosomal aneuploidy, the normal sample of chromosome.
Section partition module, the relative data amount for calculating each window obtained according to sample to be detected and normal sample
Ratio determines breakpoint location, and divides section according to breakpoint location；Wherein, the normal sample does not have chromosome aneuploidy
Property.
Section copy rate computing module is used for calculation of sector copy rate CRj。
Section aneuploidy determination module, for judging whether section has chromosome aneuploidy according to section copy rate
Property.According to Heredity theory, it is believed that 0.75<CRj<1.25 be normal, and super go beyond the scope judges j-th of section for non-multiple
Number is abnormal；If j-th of section corresponds to whole chromosome, it can determine that whether the whole chromosome is that non-integral multiple is abnormal.
In one example, in the section partition module, the relative data amount ratio is calculated using following equation：
ri=Wi/N
WiExpression falls into the ordered sequence number of i-th of window, and 1≤i≤S, S are window sum；
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence is surveyed for full-length genome
The sequence to the reference gene group is compared in the sequence that sequence obtains；
riIndicate the relative data amount of i-th of window；
For the relative data amount of i-th of window of sample to be detected；
For the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
In one example, the relative data amount ratio ViG/C content correction is carried out, the correction for obtaining each window is opposite
Data volume ratio Vi', correction relative data amount ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight, e ∈ [1, E], E be G/C content section total number；
KeFor fall into e-th of G/C content section all windows relative data amount ratio ViThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sample to be detectediThe mean value of value,
Vi' be i-th of window correction relative data amount ratio.
Wherein, E G/C content section be calculated according to the G/C content of the ordered sequence of each window in S window, and
It is divided into what the equal section in E interval obtained.In other words, according to the G/C content data meter of the ordered sequence fallen into each window
The average G/C content of ordered sequence in each window is calculated as window G/C content, G/C content is separated into the equal area in several intervals
Between, correspond to which G/C content section window falls into according to window G/C content.
In one example, in the section partition module, the breakpoint location refers to relative data amount ratio with aobvious
Write the boundary of two adjacent window apertures of sex differernce.
In one example, in the section partition module, the breakpoint location is determined by following method：It will be adjacent
N window is divided into m window group as window group, by S window, and m=S-n+1, m are window group sum；S is sample
The window sum of this division；N is window number contained in a window group, then utilizes the phase of each window in each window group
To data volume ratio ViNonparametric runs test is carried out, the P values of each adjacent window apertures group are calculated, if P values are less than threshold value, is judged
The intersection of corresponding adjacent window apertures group is the breakpoint location for copying number variation (CNV)；Wherein, P values are significant difference P values.
In one example, the section copy rate refers to the average value of each window relative data amount ratio in section.
In one example, the section copy rate is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, by calculating being averaged for each window relative data amount ratio in j-th of section
Value obtains.
In one example, in the section aneuploidy determination module, if 0.75<CRj<1.25, then judge j-th of area
Section is normal；If CRj≤ 0.75 or CRj>=1.25, then judge j-th of section for non-integral multiple exception.
In one example, if j-th of section corresponds to whole chromosome, it can determine that whether the whole chromosome is non-whole
Multiple is abnormal.
In one example, the detecting system further includes：It is cell separation module, cell whole genome amplification module, thin
Born of the same parents' amplified production quality Control module, library construction module, high-flux sequence module.
The cell separation module can be unicellular separation module, and dilution may be used in the unicellular separation module
The methods of method, mouth suction pipe partition method, micromanipulation, micro-dissections, fluidic cell exclusion, micro-fluidic, individual cells are carried out
Separation.Embryonic cell separation will be using the separation method of micromanipulation, micro-dissections.
The cell whole genome amplification module, may be used PEP-PCR, and DOP-PCR, OmniPlex WGA, MDA are (more
The methods of weight strand displacement amplification), the GenomePlex of commercial kit such as SigmaAldrich, Rubicon can also be used
The illustra GenomiPhi of the REPLI-g of the PicoPlex of Genomics, Qiagen, GEHealthcare etc. are complete to cell
Genome is expanded.In a kind of embodiment, the cell whole genome amplification module uses MDA amplification methods, to obtain
The amplified production of more complete, high coverage.The amplification principle of MDA is shown in attached drawing 2.
In one example, the cell amplified production quality Control module is used for the house-keeping gene using cell to the complete of cell
Genome amplification product carries out Quality Control, and judges whether Quality Control result meets preset condition, if meeting preset condition, illustrates
The whole genome amplification product is qualified amplified production.House-keeping gene include 8 genes, respectively CYB5A, PRPH,
GABARAPL2、ACTG1、NDUFA7、UQCRC1、MYC、MIF.If amplifying 6 or more in 8 house-keeping genes, grade
For A；If having amplified 4 or 5, it is rated B；If having amplified 2 or 3, it is rated C；If amplified
House-keeping gene number be less than 2, then be rated D.It can specify that D ranks are unqualified amplified production, can also provide C, D grades
It Wei not unqualified amplified production.
In a kind of a example, the library construction module is used to build the sequencing library of the qualified amplified production, institute
Stating library construction module includes：Broken submodule, connector connection submodule, mixing submodule, mix products purifying submodule,
PCR is enriched with submodule and enriched product purifies submodule.
Broken submodule obtains breakdown products for being crushed the qualified amplified production.It in one embodiment, can be with
Above-mentioned qualified amplified production is diluted 15~25 times, preferably 20 times, 1ng is then taken to be crushed using broken submodule.
Connector connects submodule and is used to add connector for the breakdown products, obtains connector breakdown products；
The connector breakdown products that submodule is used for the multiple samples of mixed in equal amounts are mixed, mix products are obtained；
Mix products purifying submodule is for purifying the mix products；
PCR enrichment submodules obtain enrichment production for being enriched with the mix products of the mix products purification blocks after purification
Object；
Enriched product purifying submodule obtains the sequencing library for purifying the enriched product.
Library is prepared using above-mentioned library construction module, it is broken from DNA, it connects and is enriched with to connector, total process only needs 3 small
When.
The high-flux sequence module obtains sequencing result for being sequenced according to the sequencing library.High pass measures
Illumina Hiseq series may be used in sequence, can also use Illumina NextSeq series, Miseq can also be used to survey
Sequence system can also use illumina MiniSeq sequencing systems, can also use Ion Torrent sequencing systems etc..
The embodiment of the present invention additionally provides one kind and being suitable for single celled chromosomal aneuploidy detection method, such as Fig. 1 institutes
Show, includes the following steps：S1, the partition window in the sequence of reference gene group obtain S window；S2, according to sample to be detected
The relative data amount ratio that each window obtained is calculated with normal sample determines breakpoint location, and according to breakpoint location dividing regions
Section；Wherein, the normal sample does not have chromosomal aneuploidy；Wherein, the normal sample does not have chromosome aneuploidy
Property；S3, calculation of sector copy rate CRj；S4, the whether specific chromosomal aneuploidy of section is judged according to section copy rate.
In one possible implementation, the relative data amount ratio is calculated using following equation：
ri=Wi/N
WiIndicate to fall into the ordered sequence number of i-th of window, 1≤i≤S,
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence is surveyed for full-length genome
The sequence to the reference gene group is compared in the sequence that sequence obtains；
riIndicate the relative data amount of i-th of window；
For the relative data amount of i-th of window of the sample to be detected；
For the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
In in one example, the relative data amount ratio ViG/C content correction is carried out, the correction phase of each window is obtained
To data volume ratio Vi', correction relative data amount ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeTo fall into all window V in e-th of G/C content sectioniThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sampleiThe mean value of value,
ViThe correction relative data amount ratio of ' i-th window.
In one example, the breakpoint location refers to two adjacent window apertures that relative data amount ratio has significant difference
Boundary.
In one example, the breakpoint location is determined by following method：N window adjacent in S window is drawn
It is divided into a window group, obtains m window group, m=S-n+1；M is window group sum；S is the window division module
The window sum of division；N is window number contained in a window group；Then utilize each window group in each window it is opposite
Data volume ratio ViNonparametric runs test is carried out, the P values of each adjacent window apertures group are calculated, if P values are less than threshold value, judgement pair
It is the breakpoint location for copying number variation (CNV) to answer the intersection of adjacent window apertures group；Wherein, P values are significant difference P values.
In one example, the section copy rate refers to the average value of each window relative data amount ratio in section.
In one example, the section copy rate CRjIt is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, CRjBy calculating the flat of each window relative data amount ratio in j-th of section
Mean value obtains.
In one example, in step S4, if 0.75<CRj<1.25, then judge that j-th of section is normal；If CRj≤
0.75 or CRj>=1.25, then judge j-th of section for non-integral multiple exception.
In one example, if j-th of section corresponds to whole chromosome, it can determine that whether the whole chromosome is non-whole
Multiple is abnormal.
In one example, described further comprising the steps of suitable for single celled chromosomal aneuploidy detection method：
Cell detaches；Cell whole genome amplification；Cell amplified production Quality Control；Library construction；High-flux sequence.
In one example, the cell amplified production Quality Control, for the house-keeping gene using cell to the full base of cell
Because group amplified production carries out Quality Control, and judge whether Quality Control result meets preset condition, if meeting preset condition, illustrates institute
It is qualified amplified production to state whole genome amplification product.
In one example, the library construction, including DNA is broken, connector connection, mix, mix products purify, PCR
Enrichment and enriched product purifying, the DNA broken refers to the broken qualified amplified production, obtains breakdown products；The connector
Connection refers to adding connector for the breakdown products, obtains connector breakdown products；The mixing refers to the multiple samples of mixed in equal amounts
The connector breakdown products, obtain mix products；The mix products purifying refers to the purifying mix products；The enrichment
Product purification refers to the purifying enriched product, obtains sequencing library.
It is provided in an embodiment of the present invention to be suitable for single celled chromosomal aneuploidy detecting system and method, have as follows
Advantageous effect：
(1), required sample initial amount is low, and the chromosomal aneuploidy of detection individual cells may be implemented.
(2), window size can be set according to demand, and the size by controlling window improves detection resolution.
(3), whole genome amplification product Quality Control
After embryonic cell completes single cell whole genome amplification, Quality Control will be carried out to amplified production, to evaluate full genome
Whether group amplification procedure succeeds, such as unsuccessful, then terminates experiment, reduce cost consumption.
(4), it is enriched with after first sample mixing
During building library, mixed in equal amounts first is carried out to all sample DNAs after jointing, then carries out PCR enrichments,
The data volume of each sample output can be made more uniform using this method, avoid unnecessary data from wasting, and can subtract
Cost consumption after few enrichment experiment, reduces operating procedure, reduces operation complexity.
(5), 1ng originates DNA and builds library
Cell sample belongs to trace sample, and amount of DNA is few, and detection can be completed in entire detection process, 1ng DNA, reduces
The waste of sample.
(6), the reads numbers of 700k can complete the variation detection of 5Mb or more
Use most 700k reads/ samples, you can chromosome deficiency/repetition to the pattern detection 5Mb or more, it is accurate
True rate is up to 99.9%.Sequencing depth is reduced, cost is reduced.
(7), sample data is corrected
Because cell sample passes through whole genome amplification, many amplification deviations can be introduced, this method introduces sample data correction,
Reduce influence of these deviations to result.
Next it is suitable for single celled chromosome aneuploidy to provided in an embodiment of the present invention in a specific example
Property detection method carries out citing introduction.
Embodiment 1
Sample is chosen as follows：
Normal man's leucocyte, is denoted as male；Normal female's leucocyte, is denoted as female；Normal embryonic cells are denoted as EC-1；
T22 exceptions embryonic cell (T22 is the abbreviation of trisomy 22, indicates No. 22 chromosome trisomies), is denoted as EC-2；Segment
Missing/repetition embryonic cell (q25.1-q26 repetitions), is denoted as EC-3.
It should be noted that in embodiments of the present invention, " normal " indicates that its cell does not have chromosomal aneuploidy,
That is the chromosome in cell is all integral multiple.
Experimental implementation flow is as follows：
1, embryonic cell detaches
Under the microscope, 3-10 cell of embryonic trophoblasts cell cutting is inhaled using capillary using laser cutting technique
Pipe, which is inhaled, all takes out the cell under cutting, is subsequently placed in PBS and cleans three times, and is put into the PCR pipe of 3.5 μ l PBS and centrifuges
It is spare.
2, single cell whole genome amplification
The REPLI-g single cell kit kits of Qiagen are selected to carry out single cell whole genome amplification, specifically
Operation is as follows：
21, match liquid：A new pipe DLB freeze-dried powders：Add 500 μ lddH2O dissolves；D2Buffer is formulated as follows：
It is no more than one month in -20 DEG C of storages, resting period after mixing.
22, it is operated on ice chest, is added in 3 μ lD2Buffer to 4 μ l PBS and (includes cell sample), flick mixing, 65 DEG C
It is incubated 10min.
23, it is operated on ice chest, adds 3 μ l Stop Solution, flick mixing, be placed in 4 DEG C of refrigerator coolings.
24, match MDA mixed liquors, shake mixing；MDA Compound mixed solutions are as follows：
25, the MDA mixed liquors of 40 μ l steps 24 preparation are added in the mixed liquor in step 23, shake mixing.
26, heat is arranged on thermocycler to cover and execute following procedure：
30℃ 8h
65℃ 3min
4℃ ∞
27, the product of step 26 is put in (long-term preservation is in -20 DEG C) spare at 4 DEG C after rapid centrifugation.
3, unicellular amplified production Quality Control
Select 8 pairs of genomes on house-keeping gene (CYB5A, PRPH, GABARAPL2, ACTG1, NDUFA7, UQCRC1,
MYC, MIF), Quality Control is carried out to the product of step 26, to determine whether amplification procedure is qualified.Operating process is as follows：
It should be noted that the product of step 26 is unicellular amplified production, it is referred to as WGA products.
31, by 30 times of WGA product dilutions, and according to following system configurations PCR mixed liquors：
32, the WGA products after 1 μ l dilution are taken, 19 μ l PCR mixed liquors are separately added into, use pressure-vaccum above and below pipettor 10 times
Mixing, or concussion mixing 10s on the oscillator；
33, of short duration centrifugation, is put on thermocycler, heat lid is arranged, and run following procedure:
34,10 μ l PCR products are taken, electrophoresis detection is carried out using 2% Ago-Gel, uses 1kb Marker.
35, quality control standard：
4, library construction：
41, WGA products are broken
411, by 20 times of WGA product dilutions after being diluted in step 31, library process is built after taking 1ng to carry out respectively.
412, heat is arranged on thermocycler to cover and following procedure, program nomenclature Frag is arranged：
Program is run, and is pause running to 4 DEG C.
413, according to following system by required reagent mixing in 0.2mlPCR pipes, DNA sample amount is insufficient, is mended with sterile water
Foot.
Step 413 operates on ice chest, 5 х WGS Fragmentation Mix using preceding need fully shaking mixing 30s with
On.
414, using 10 mixings of pressure-vaccum above and below pipettor, or concussion mixing 10s (notes on the oscillator：Ensure that often pipe is anti-
Liquid is answered to be in melting state)；
415, of short duration centrifugation, is put on thermocycler, executes Frag programs.
42, connector connects
421, following reagent broken sample is directly added into mix in liquid：
422, using 10 mixings of pressure-vaccum above and below pipettor, or concussion mixing 10s on the oscillator；
423, of short duration centrifugation, 25 DEG C of hatching 10min on thermocycler；
424, rapid centrifugation takes 2.5 μ l products to be mixed into new PCR pipe in each reaction tube.
425, mixing, of short duration centrifugation are shaken, mixed volume is 12.5 μ l (3+2 check samples of embryo's sample)【It needs
It is bright, herein mixed in equal amounts is carried out for each sample DNA after connecting connector】.
43, product mixing purifying
431, shift to an earlier date that half an hour, AMPure XP Beads were resuspended and to be put in room temperature spare, and configure 80% alcohol (per instead
Answer 1000 μ l) it is spare；
432, it is added in the connection mix products of the μ l of AMPure XP Beads to 12.5 after 8 μ l are resuspended, on liquid-transfering gun
Lower pressure-vaccum mixing 10 times or oscillator oscillation mixing 10s；
433, it is placed at room temperature for 5min；
434, after rapid centrifugation, it is placed in 5min on magnetic frame, until solution is clarified, supernatant is carefully removed, not throw away
Magnetic bead；
435, magnetic frame remains stationary as, and 80% alcohol of the 200 fresh configurations of μ l is added into centrifuge tube, is placed at room temperature for 30s,
It is careful to remove supernatant；
436,435 steps are repeated；
437, centrifuge tube is maintained on magnetic frame and opens centrifuge tube lid, is air-dried about 5min (notes：Magnetic bead can not be excessive
It is dry)
438, centrifuge tube is removed from magnetic frame, is added the distilled water of 52 μ l, liquid-transfering gun up and down pressure-vaccum to magnetic bead is resuspended,
It is placed at room temperature for 5min；
439, rapid centrifugation, and centrifuge tube is placed on magnetic frame, it is placed at room temperature for about 5min, until solution is clarified, shifts 50 μ
In l supernatants to new PCR pipe；
4310, it is added in the μ l purified products of AMPure XP Beads to 50 after 40 μ l are resuspended, with pressure-vaccum above and below liquid-transfering gun
Mixing 10 times or oscillator oscillation mixing 10s；
4311,433-437 steps are repeated；
4312, centrifuge tube is removed from magnetic frame, the distilled water of 22 μ l, liquid-transfering gun 10 extremely resuspensions of pressure-vaccum up and down is added
Magnetic bead is placed at room temperature for 5min；
4313, rapid centrifugation, and centrifuge tube is placed on magnetic frame, it is placed at room temperature for about 5min, until solution is clarified, transfer 20
In μ l supernatants to new PCR pipe, linker DNA is obtained.
44, PCR is enriched with
441, following reagent is added in the linker DNA that step 4313 obtains：
442, using 10 mixings of pressure-vaccum above and below pipettor, or concussion mixing 10s on the oscillator；
443, of short duration centrifugation, is put on thermocycler, heat lid is arranged, and run following procedure：
45, enriched product purifies
451, shift to an earlier date that half an hour, AMPure XP Beads were resuspended and to be put in room temperature spare, and configure 80% alcohol (per instead
Answer 500 μ l) it is spare；
452, it is added in the PCR product of the μ l of AMPure XP Beads to 50 after 45 μ l are resuspended, with pressure-vaccum above and below liquid-transfering gun
Mixing 10 times or oscillator oscillation mixing 10s；
453, it is placed at room temperature for 5min；
454, after rapid centrifugation, it is placed in 5min on magnetic frame, until solution is clarified, supernatant is carefully removed, not throw away
Magnetic bead；
455, magnetic frame remains stationary as, and 80% alcohol of the 200 fresh configurations of μ l is added into centrifuge tube, is placed at room temperature for 30s,
It is careful to remove supernatant；
456,455 steps are repeated；
457, centrifuge tube is maintained on magnetic frame and opens centrifuge tube lid, is air-dried about 5min (notes：Magnetic bead can not be excessive
It is dry)
458, centrifuge tube is removed from magnetic frame, the 1 х TE Buffer of 22 μ l, liquid-transfering gun pressure-vaccum 10 times up and down is added
To magnetic bead is resuspended, it is placed at room temperature for 5min；
459, rapid centrifugation, and centrifuge tube is placed on magnetic frame, it is placed at room temperature for about 5min, until solution is clarified, shifts 20 μ
L supernatants are to new centrifuge tube.
Obtain library A.
4510, quantitative and fragment analysis is carried out (referring to phase using Qubit 3.0 and agilent 2100bioanalyzer
Close product description).
5, high-flux sequence
The embodiment of the present invention carries out high-flux sequence using Illumina MiSeq sequencing systems to library A.It prepares
Library MiSeq sequenators operation, 2 × 75bp of sequencing length.
Herein, it should be noted that during the present inventor also establishes library, the time of sample DNA mixing
Sequence has made comparison sequencing.As described above, library A is the progress mixed in equal amounts after sample DNA jointing, text is then formed
Library A.Another method is respectively to build library by not mixed directly after each sample DNA jointing, then will
Each sample library is mixed, and library B is obtained.Library A and library B is sequenced on same sequenator, the data of acquisition
The CV of yield is respectively 15.5% and 32.6%.It can be seen that carry out mixed in equal amounts after sample DNA jointing, then shape
At library A, the data volume of each sample output can be made more uniform, avoid unnecessary data from wasting, and can reduce
Cost consumption after enrichment experiment reduces operating procedure, reduces operation complexity.
6, comparing is to reference gene group
By the read comparings obtained to library A sequencings to reference gene group, it is reference gene group sequence to select people HG19
Row, and comparison result is counted.The statistical result of comparing result is as shown in table 1.
Table 1
Wherein Total read indicate the total read numbers of each sample；Mappable read expressions can compare reference
The read numbers of genome；Map rate indicate comparison rate；Unique Read indicate unique read compared to reference gene group
Number；Unique Rate indicate unique comparison rate, i.e., unique to compare the ratio that the read that compared is accounted for the read of reference gene group
Rate.
7, Counting statistics amount
To the sequencing result of library A：
s1：Partition window
The partition window in the sequence of reference gene group obtains S window.There are many optional modes for partition window, one
It, can be according to the equal partition window of base number of reference gene group in each window in kind embodiment；In another embodiment
In, the equal model split window of the ordered sequence number of each window can be fallen into the sequence of normal sample；Implement in another kind
In mode, the equal model split window of the ordered sequence number of each window can be fallen into the simulated series of reference gene group.It is single
Base number or the ordered sequence number fallen in single window can be set as required in a window, such as can take 50-
Any number in 1000kb, specifically, for example choosing 500kb, digital smaller under normal circumstances, as a result resolution ratio is higher.
s2：Calculate the relative data amount r of each windowi
The ordered sequence for counting each sample falls into the relative data amount r of each windowi,
ri=Wi/N
WiIndicate to fall into the ordered sequence number of i-th of window, 1≤i≤S,
N indicates the ordered sequence number sum that sample sequencing obtains；
riIndicate the relative data amount of i-th of window；
s3：Calculate the relative data amount ratio V of each windowi
Using normal sample as normal control, counts sample to be detected and fall into the opposite of each window with normal control sample
Data volume ratio Vi,
For the relative data amount of i-th of window of sample to be detected；
For the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window；
s4：Breakpoint location is determined to divide section：
Using n adjacent window as window group, S window is divided into m window group, m=S-n+1
M is window group sum；
S is the window sum that sample divides；
N is window number contained in a window group；
Utilize the relative data amount ratio V of each window in each window groupiNonparametric runs test is carried out, is calculated each adjacent
The P values (significant difference P values) of window group, if P values be less than threshold value (threshold value can be empirical value, such as 0.001；It can also lead to
Cross the acquisition of the analysis to multiple normal samples), then judge the intersection of corresponding adjacent window apertures group for copy number variation (CNV)
Breakpoint location.By upper b breakpoint of acquisition, reference gene group sequence is divided by b+1 section with breakpoint.Wherein, adjacent window apertures
Group refers to the adjacent window group of head and the tail, and each two adjacent window apertures group corresponds to a P value.
s5：Calculate the copy rate CR of each sectionj
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section；
CRjBy calculating each window relative data amount ratio V in j-th of sectioniAverage value obtain.
s6：Judge whether section is that non-integral multiple is abnormal
According to Heredity theory, it is believed that 0.75<CRj<1.25 be normal, and super go beyond the scope judges that j-th of section is
Non- integral multiple is abnormal.If j-th of section corresponds to whole chromosome, it can determine that whether the whole chromosome is that non-integral multiple is different
Often.
It further preferably,, can be to the relative data amount ratio V of each window in order to improve the accuracy rate of data in step S3i
G/C content correction is carried out, the correction relative data amount ratio V of each window is obtainedi′。
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeTo fall into all windows (number of windows is indicated with g) relative data amount ratio V in e-th of G/C content sectioniIt is equal
Value,
For all window relative data amount ratio V of sampleiMean value,
Vi' be i-th of window correction relative data amount ratio,
The average GC that ordered sequence in each window is calculated according to the G/C content data of the ordered sequence fallen into each window contains
Amount is used as window G/C content, and G/C content is separated into the equal section in several intervals, if for example, can be divided into for interval with 0.01
Dry section, corresponds to which G/C content section window falls into according to window G/C content.
In one embodiment, as shown in figure 3, taking each window G/C content and relative data amount ratio ViMapping, by GC
Content is divided into several sections with 0.01 for interval, obtains the window for falling into each G/C content section, according to falling into corresponding G/C content area
Between window relative data amount ratio Vi, the correction relative data amount ratio for obtaining each window can be calculated using above-mentioned formula
Vi′。
When to the relative data amount ratio V of each windowiAfter having carried out G/C content correction, the results are shown in Figure 4.Step s4
In, utilize the correction relative data amount ratio V of each window in each window groupi' carry out nonparametric runs test；
In one embodiment, to the relative data amount ratio V of each window in Fig. 3iAfter having carried out G/C content correction,
Utilize the correction relative data amount ratio V of each window in each window groupi' carry out nonparametric runs test.
In step s5, copy rate calculation formula is：
H is the window number in j sections；
B is breakpoint number；
CRj ' is the copy rate of j-th of section.
CRj ' is obtained by calculating the average value of each window relative data amount ratio in j-th of section.
In step s6, according to Heredity theory, it is believed that 0.75<CRj′<1.25 be normal, and super go beyond the scope judges the
J section is that non-integral multiple is abnormal.If j-th of section corresponds to whole chromosome, it can determine that whether the whole chromosome is non-
Integral multiple is abnormal.
According to above-mentioned judgment method, the chromosome of T22 exception embryonic cells is as shown in Figure 5；Fragment deletion/repetition embryo is thin
The chromosome of born of the same parents is as shown in Figure 6；The chromosome of normal embryonic cells is as shown in Figure 7.
The result of Fig. 5, Fig. 6 and Fig. 7 absolutely prove：The detecting system can correctly distinguish normal embryo and abnormal embryo
Tire.
Scheme using the present invention, required original samples are less, and detection can be completed in 1ngDNA, can be used for rare sample
Detection；After the extension of cell full-length genome, Quality Control is carried out to amplified production, it is whether qualified to evaluate whole genome amplification result,
If unqualified, experiment is terminated, to reduce unnecessary testing cost.
To sum up technical scheme of the present invention has the following advantages that：
(1), required sample initial amount is low, and the chromosomal aneuploidy of detection individual cells may be implemented.(2), Ke Yigen
Window size is set according to demand, and the size by controlling window improves detection resolution.(3), whole genome amplification product matter
Control will carry out Quality Control, to evaluate whole genome amplification after embryonic cell completes single cell whole genome amplification to amplified production
Whether process succeeds, such as unsuccessful, then terminates experiment, reduce cost consumption.(4), it is enriched with after first sample mixing, during building library,
Mixed in equal amounts first is carried out to all sample DNAs after jointing, PCR enrichments is then carried out, can be made using this method each
The data volume of sample output is more uniform, avoids unnecessary data from wasting, and can reduce the cost after enrichment experiment
Consumption reduces operating procedure, reduces operation complexity.(5), 1ng originates DNA and builds library, and cell sample belongs to trace sample, DNA
Amount is few, and detection can be completed in entire detection process, 1ng DNA, reduces the waste of sample.(6), the reads numbers of 700k can be complete
It is detected at the variation of 5Mb or more, uses most 700k reads/ samples, you can to the chromosome of the pattern detection 5Mb or more
Missing/repetition, accuracy rate is up to 99.9%.Sequencing depth is reduced, cost is reduced.(7), sample data is corrected, because of cell sample
By whole genome amplification, many amplification deviations can be introduced, this method introduces sample data correction, reduces these deviations to result
Influence.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology can all carry out modifications and changes to above-described embodiment without violating the spirit and scope of the present invention.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should by the present invention claim be covered.
Claims (24)
1. one kind being suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described suitable for single celled
Chromosomal aneuploidy system includes：
Window division module, for the partition window in the sequence of reference gene group；
Section partition module, the relative data amount ratio for calculating each window obtained according to sample to be detected and normal sample
It determines breakpoint location, and section is divided according to breakpoint location；Wherein, the normal sample does not have chromosomal aneuploidy；
Section copy rate computing module is used for calculation of sector copy rate CRj；
Section aneuploidy determination module, for judging whether section has chromosomal aneuploidy according to section copy rate.
2. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
In section partition module, the relative data amount ratio is calculated using following equation：
ri=Wi/N
Vi=ri case/ri control
WiExpression falls into the ordered sequence number of i-th of window, and 1≤i≤S, S are window sum；
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence obtains for genome sequencing
The sequence to the reference gene group is compared in the sequence obtained；
riIndicate the relative data amount of i-th of window；
ri caseFor the relative data amount of i-th of window of sample to be detected；
ri controlFor the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
3. according to claim 2 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
Relative data amount ratio ViG/C content correction is carried out, the correction relative data amount ratio V of each window is obtainedi', correct relative data
Measure ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeFor fall into e-th of G/C content section all windows relative data amount ratio ViThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sample to be detectediThe mean value of value,
Vi' be i-th of window correction relative data amount ratio.
4. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
In section partition module, the breakpoint location refers to the friendship for two adjacent window apertures that relative data amount ratio has significant difference
Boundary.
5. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
In section partition module, the breakpoint location is determined by following method：
Using n adjacent window as window group, S window is divided into m window group, m=S-n+1,
M is window group sum；
S is the window sum that sample divides；
N is window number contained in a window group, then utilizes the relative data amount ratio V of each window in each window groupi
Nonparametric runs test is carried out, the P values of each adjacent window apertures group are calculated, if P values are less than threshold value, judges corresponding adjacent window apertures group
The intersection of body is the breakpoint location for copying number variation (CNV)；Wherein, P values are significant difference P values.
6. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
Section copy rate refers to each window relative data amount ratio V in sectioniAverage value.
7. according to claim 6 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
Section copy rate is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, the average value by calculating each window relative data amount ratio in j-th of section obtains
.
8. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that described
In section aneuploidy determination module, if 0.75<CRj<1.25, then judge that j-th of section is normal；If CRj≤ 0.75 or CRj
>=1.25, then judge j-th of section for non-integral multiple exception.
9. according to claim 8 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that if the
J section corresponds to whole chromosome, then can determine that whether the whole chromosome is that non-integral multiple is abnormal.
10. according to claim 1 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that institute
Stating detecting system further includes：Cell separation module, cell whole genome amplification module, cell amplified production quality Control module, library
Build module, high-flux sequence module.
11. according to claim 10 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that institute
It states cell amplified production quality Control module and Quality Control is carried out to the whole genome amplification product of cell for the house-keeping gene using cell,
And judge whether Quality Control result meets preset condition, if meeting preset condition, illustrate that the whole genome amplification product is
Qualified amplified production.
12. according to claim 10 be suitable for single celled chromosomal aneuploidy detecting system, which is characterized in that institute
Sequencing library of the library construction module for building the qualified amplified production is stated, the library construction module includes：Broken son
Module, connector connection submodule, mixing submodule, mix products purifying submodule, PCR enrichment submodules and enriched product purifying
Submodule, broken submodule obtain breakdown products for being crushed the qualified amplified production；The connector connection submodule is used for
Connector is added for the breakdown products, obtains connector breakdown products；The mixing submodule is for the multiple samples of mixed in equal amounts
The connector breakdown products, obtain mix products；The mix products purifying submodule is for purifying the mix products；It is described
PCR enrichment submodules obtain enriched product for being enriched with the mix products of the mix products purification blocks after purification；The richness
Collection product purification submodule obtains the sequencing library for purifying the enriched product.
13. one kind being suitable for single celled chromosomal aneuploidy detection method, which is characterized in that include the following steps：
S1, the partition window in the sequence of reference gene group obtain S window；
S2, the relative data amount ratio that each window obtained is calculated according to sample to be detected and normal sample determine breakpoint location,
And section is divided according to breakpoint location；Wherein, the normal sample does not have chromosomal aneuploidy；Wherein, the normal sample
This does not have chromosomal aneuploidy；
S3, calculation of sector copy rate CRj；
S4, judge whether section has chromosomal aneuploidy according to section copy rate.
14. according to claim 13 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
Relative data amount ratio is stated to calculate using following equation：
ri=Wi/N
Vi=ri case/ri control
WiIndicate to fall into the ordered sequence number of i-th of window, 1≤i≤S,
N indicates the ordered sequence number sum that sample sequencing to be detected obtains；Wherein, the ordered sequence obtains for genome sequencing
The sequence to the reference gene group is compared in the sequence obtained；
riIndicate the relative data amount of i-th of window；
ri caseFor the relative data amount of i-th of window of the sample to be detected；
ri controlFor the relative data amount of i-th of window of normal sample；
ViFor the relative data amount ratio of i-th of window.
15. according to claim 14 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that
The relative data amount ratio ViG/C content correction is carried out, the correction relative data amount ratio V of each window is obtainedi', correction
Relative data amount ratio Vi' calculated using following equation：
Vi'=Vi/Ce
CeFor fall into e-th of G/C content section window weight,
KeTo fall into all window V in e-th of G/C content sectioniThe mean value of value,
G is all windows sum for falling into e-th of G/C content section,
For the relative data amount ratio V of all windows of sampleiThe mean value of value,
Vi' be i-th of window correction relative data amount ratio.
16. according to claim 13 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
State the boundary that breakpoint location refers to two adjacent window apertures that relative data amount ratio has significant difference.
17. according to claim 16 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
Breakpoint location is stated to determine by following method：
N window adjacent in S window is divided into a window group, obtains m window group, m=S-n+1；M is window
Mouth group sum；S is the window sum that the window division module divides；N is window number contained in a window group；
Then the relative data amount ratio V of each window in each window group is utilizediNonparametric runs test is carried out, is calculated each adjacent
The P values of window group judge the intersection of corresponding adjacent window apertures group for copy number variation (CNV) if P values are less than threshold value
Breakpoint location；Wherein, P values are significant difference P values.
18. according to claim 13 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
Stating section copy rate refers to, the average value of each window relative data amount ratio in section.
19. according to claim 18 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that
The section copy rate CRjIt is calculated using following equation：
H is the window number in j sections；
B is breakpoint number；
CRjFor the copy rate of j-th of section, CRjBy calculating each window relative data amount ratio V in j-th of sectioniBe averaged
Value obtains.
20. according to claim 13 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that step
In rapid S4, if 0.75<CRj<1.25, then judge that j-th of section is normal；If CRj≤ 0.75 or CRj>=1.25, then judge jth
A section is that non-integral multiple is abnormal.
21. according to claim 20 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that if
J-th of section corresponds to whole chromosome, then can determine that whether the whole chromosome is that non-integral multiple is abnormal.
22. according to claim 13 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
It states further comprising the steps of suitable for single celled chromosomal aneuploidy detection method：Cell detaches；Cell full-length genome expands
Increase；Cell amplified production Quality Control；Library construction；High-flux sequence.
23. according to claim 22 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
Cell amplified production Quality Control is stated, Quality Control is carried out to the whole genome amplification product of cell for the house-keeping gene using cell, and
Judge whether Quality Control result meets preset condition, if meeting preset condition, illustrates that the whole genome amplification product is to close
Lattice amplified production.
24. according to claim 22 be suitable for single celled chromosomal aneuploidy detection method, which is characterized in that institute
Library construction is stated, including DNA is broken, connector connection, mixing, mix products purifying, PCR is enriched with and enriched product purifying, it is described
DNA broken refers to the broken qualified amplified production, obtains breakdown products；The connector connection refers to adding for the breakdown products
Adjunction head obtains connector breakdown products；The mixing refers to the connector breakdown products of the multiple samples of mixed in equal amounts, is mixed
Close product；The mix products purifying refers to the purifying mix products；The enriched product purifying refers to the purifying enrichment
Product obtains sequencing library.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810078283.XA CN108363903B (en) | 2018-01-23 | 2018-01-23 | Chromosome aneuploidy detection system suitable for single cell and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810078283.XA CN108363903B (en) | 2018-01-23 | 2018-01-23 | Chromosome aneuploidy detection system suitable for single cell and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108363903A true CN108363903A (en) | 2018-08-03 |
| CN108363903B CN108363903B (en) | 2022-03-04 |
Family
ID=63006985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810078283.XA Active CN108363903B (en) | 2018-01-23 | 2018-01-23 | Chromosome aneuploidy detection system suitable for single cell and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108363903B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111226281A (en) * | 2019-12-31 | 2020-06-02 | 深圳华大临床检验中心 | Method and device for determining chromosome aneuploidy and constructing classification model |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013059967A1 (en) * | 2011-10-28 | 2013-05-02 | 深圳华大基因科技有限公司 | Method for detecting micro-deletion and micro-repetition of chromosome |
| US20150066376A1 (en) * | 2013-09-03 | 2015-03-05 | Welgene Biotech Co., Ltd. | Non-invasive prenatal testing method based on whole-genome tendency scoring |
| CN104520437A (en) * | 2013-07-17 | 2015-04-15 | 深圳华大基因科技有限公司 | Method and device for detecting chromosomal aneuploidy |
| CN106520940A (en) * | 2016-11-04 | 2017-03-22 | 深圳华大基因研究院 | Chromosomal aneuploid and copy number variation detecting method and application thereof |
-
2018
- 2018-01-23 CN CN201810078283.XA patent/CN108363903B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013059967A1 (en) * | 2011-10-28 | 2013-05-02 | 深圳华大基因科技有限公司 | Method for detecting micro-deletion and micro-repetition of chromosome |
| CN104520437A (en) * | 2013-07-17 | 2015-04-15 | 深圳华大基因科技有限公司 | Method and device for detecting chromosomal aneuploidy |
| US20150066376A1 (en) * | 2013-09-03 | 2015-03-05 | Welgene Biotech Co., Ltd. | Non-invasive prenatal testing method based on whole-genome tendency scoring |
| CN106520940A (en) * | 2016-11-04 | 2017-03-22 | 深圳华大基因研究院 | Chromosomal aneuploid and copy number variation detecting method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111226281A (en) * | 2019-12-31 | 2020-06-02 | 深圳华大临床检验中心 | Method and device for determining chromosome aneuploidy and constructing classification model |
| CN111226281B (en) * | 2019-12-31 | 2023-03-21 | 深圳华大医学检验实验室 | Method and device for determining chromosome aneuploidy and constructing classification model |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108363903B (en) | 2022-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Farlik et al. | DNA methylation dynamics of human hematopoietic stem cell differentiation | |
| CN102329876B (en) | Method for measuring nucleotide sequence of disease associated nucleic acid molecules in sample to be detected | |
| KR101850437B1 (en) | Method for predicting transplantation rejection using next generation sequencing | |
| JP6830094B2 (en) | Nucleic acids and methods for detecting chromosomal abnormalities | |
| DK3078752T3 (en) | SOLUTION OF REFRACTIONS USING POLYMORPHISM COUNTIES | |
| KR20180123020A (en) | Use of DNA fragment size to determine copy number variation | |
| JP2015534807A (en) | Non-invasive method for detecting fetal chromosomal aneuploidy | |
| TW201122470A (en) | Fetal genomic analysis from a maternal biological sample | |
| CN109136364A (en) | Pass through extensive parallel RNA sequencing analysis mother's blood plasma transcript profile | |
| US20200407806A1 (en) | Snp molecular marker tightly linked to weeping trait of mei and detection method and use thereof | |
| Yahagi et al. | Position‐specific expression of Hox genes along the gastrointestinal tract | |
| KR20210039406A (en) | Cell-free DNA damage analysis and clinical application thereof | |
| Rodriguez et al. | Genetic variation in the immunoglobulin heavy chain locus shapes the human antibody repertoire | |
| Olsson et al. | Genome-wide analyses suggest mechanisms involving early B-cell development in canine IgA deficiency | |
| CN104388551A (en) | Kit for detecting mutation of Von Hippel-Lindau (VHL) syndrome related gene | |
| CN105209642A (en) | Methods of genome sequencing and epigenetic analysis | |
| CN108363903A (en) | One kind being suitable for single celled chromosomal aneuploidy detecting system and application | |
| CN115216545A (en) | Method for noninvasive pre-implantation aneuploidy genetic detection | |
| KR20220118295A (en) | High Throughput Single Cell Libraries, and Methods of Making and Using the Same | |
| CN109055547B (en) | Biomarker for evaluating aortic dissection risk and application thereof | |
| Felice et al. | Prenatal diagnosis by whole exome sequencing in fetuses with ultrasound abnormalities | |
| Rosenfeld et al. | Bulk gDNA sequencing of antibody heavy-chain gene rearrangements for detection and analysis of B-cell clone distribution: A method by the AIRR community | |
| Priya et al. | Exome sequencing: capture and sequencing of all human coding regions for disease gene discovery | |
| WO2022015998A1 (en) | Gene panels and methods of use thereof for screening and diagnosis of congenital heart defects and diseases | |
| WO2023077487A1 (en) | Adenovirus mnp marker combination, primer pair combination, kit and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A chromosomal aneuploidy detection system suitable for single cells and its application Effective date of registration: 20231201 Granted publication date: 20220304 Pledgee: Industrial Bank Co.,Ltd. Shanghai Changning sub branch Pledgor: BASETRA MEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2023310000796 |