CN107868830A - A set of SNP site for canine ore grade indexes - Google Patents

A set of SNP site for canine ore grade indexes Download PDF

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Publication number
CN107868830A
CN107868830A CN201711109552.6A CN201711109552A CN107868830A CN 107868830 A CN107868830 A CN 107868830A CN 201711109552 A CN201711109552 A CN 201711109552A CN 107868830 A CN107868830 A CN 107868830A
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canine
snp site
chip
ore grade
snp
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CN107868830B (en
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刘毓文
杨承
耿灿
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Shenzhen Deep Known Biotechnology Co Ltd
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    • 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
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    • 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
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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a set of SNP site for canine ore grade indexes, the SNP site for canine ore grade indexes includes giving SNP site in table 2.The invention also discloses a kind of chip for canine ore grade indexes, the chip is used in measurement table 2 allele for giving SNP site.

Description

A set of SNP site for canine ore grade indexes
Technical field
The invention belongs to field of gene detection, more particularly it relates to a set of SNP for canine ore grade indexes Site.
Background technology
The method of canine genetic strain identification is broadly divided into three major types at present.The first kind：By noting down canine man on record Spectrum information judges the strain of canine.Second class：Strain judgement is carried out by canine appearance character.3rd class：Pass through DNA microsatellites (STR) is judged strain in site.
The technical disadvantages of above-mentioned three classes product are as follows：The first kind：1) canine is needed to be always maintained at systematic family tree file, If file is lost, or file is tampered (bad pet dealer in fraud of consumer often with this means), and pet strain is sentenced It is disconnected to will appear from mistake.2) in the market circulates, or the miscegenation pet kind of dog of pet owner raising, the overwhelming majority do not have system Family tree file, so at all can not accurate judgement its strain composition.Second class：1) by pet outward appearance, it is difficult to distinguish product It is close dog strains.2) judgement is very subjective, and can not provide objective evidence support.3) can not rational judgment go out the purity of strain It is how high.4) if mixed-blood kind, several can not form from outward appearance accurate judgement strain.3rd class：1) because STR marker bits Point quantity is few, the accurate canine database judged dependent on enormous amount.2) experiment flow is difficult to facilitate compatibility to enter the sequencing of two generations Platform, can not be effectively combined into other disease detections being sequenced with two generations based on DNA and trait predictive one it is integrated Experimental method.3) because STR marker site quantity is few, information content is limited, and this method has acceptable point to purebred blood lineage Resolution, but can not make accurate judgement for the canine of more lineage promiscuities.
The content of the invention
We have developed computerized algorithm, and most representative 500 are have selected from 230,000 DNA SNP markers of canine Site, the strain for judging canine with the DNA data in this 500 sites form.
Therefore, in a first aspect, the invention provides a set of SNP site for canine ore grade indexes, the SNP site Including giving SNP site in table 2.
In one embodiment, the SNP site includes giving SNP site in table 1.
In one embodiment, the canine strain includes Miniature Schnauzer, Ke Li, Pembroke Welsh Corgi And Bichon Frise.
Compared with existing scheme one and scheme two, we carry out strain with the DNA information in saliva or blood and judged.With Existing scheme three is compared, and what we used is SNP (the unit point nucleotide polymorphisms for being capable of the inspection of convenient use two generations microarray dataset Property).Compared with scheme four, the number of sites that we use is less.
Brief description of the drawings
By the following drawings, the present invention will be described
Fig. 1 is the qualification result of Miniature Schnauzer, and left column is the analysis only made of 500 SNP sites, and the right side is classified as full genome The analysis that 230,000 sites of group are done.
Fig. 2 is Ke Li qualification result, and left column is the analysis only made of 500 SNP sites, and the right side is classified as full-length genome 230,000 The analysis that individual site is done.
Fig. 3 is the qualification result of Pembroke Welsh Corgi, and left column is the analysis only made of 500 SNP sites, right It is classified as the analysis that 230,000 sites of full-length genome are done.
Fig. 4 is the qualification result of Bichon Frise, and left column is the analysis only made of 500 SNP sites, and the right side is classified as full genome The analysis that 230,000 sites of group are done.
Embodiment
Compared with existing scheme one, 1) carry out strain with the DNA information in saliva or blood to judge, the DNA of canine can To be extracted at any time from canine, Pedigree Documents are prevented and have lost the risk for leading to not carry out strain judgement.2) DNA reads Information Center It is objective to learn, and can be operated by canine buyer by third-party platform, it is therefore prevented that canine dealer distorts pedigree information Cheat the possibility of consumer.3) canine that mixed-blood strain does not have pedigree information can also be judged strain composition.With it is existing Scheme two is compared, the ore grade indexes using DNA data as foundation, and science is accurately objective.Although similar kind of dog outward appearance is close, Difference in DNA aspects can pass through our algorithm accurate judgement.Our algorithm can also be to mixed-blood dog only each strain Component accurate judgement.Compared with existing scheme three, that we use is the SNP for being capable of the inspection of convenient use two generations microarray dataset (unit point nucleotide polymorphisms), meanwhile, the far super STR of bit number of points of our Algorithm Analysis, therefore mixed-blood dog only can also be cooked To accurate ore grade indexes.Compared with existing scheme four, the site that we use is few, while is enough to carry out accurately strain again Judge, thus can low cost carry out conduct identification.
The statistic algorithm developed with us selects 500 canine SNP sites.With our analyzing software system to pet Strain is identified.
In the present invention, dog genome, which is derived under CanFam3.1., gives chromosome numbers and SNP site (being shown in Table 1).
Table 1
1：ID, this ID are the code names of canine SNP site；2：Chromosome；3：Position, numeral represent position on chromosome Put；4：Ke Li；5：Miniature Schnauzer；6：Compare bear；7：Ke Ji；Two characters in 4-7 represent the gene on each line position point Type, " 0 " represent that site information is not reaching to quality control standard, are not used in analysis.Chip platform has some site primers It is not up to standard, but as long as ratio is little, would not have a significant impact to accurate judged result.
Inventor is screened to have obtained above-mentioned 500 SNP sites to 230,000 DNA SNP marker information of canine, is passed through The canine DNA data of this 500 SNP sites judge the strain composition of canine.
The system of selection of traditional ancestral system informative site mark is based on genetic differentiation coefficient Fst.This method is based on Hardy Weinberg equilibrium model to each site, it is necessary to calculate the statistic between every a pair of strains, and then synthesis uses each pair strain Result of calculation.For more than number and inorganic strain (the artificial strain of kind more than 200), this model hypothesis is untenable. But we regard the analysis of purebred strain as the classification problem in one machine learning, the non-model method of machine learning is borrowed, The feasibility and representativeness of classification problem feature selection approach known to investigating and weighing.Due to needing to consider under many kinds of parameters Feature selecting scheme, we use the faster SVM and CNN of arithmetic speed relative degrees mixed model as feature selection approach Judgment criteria.
Table 2 (distinguishes Miniature Schnauzer, Ke Li, Pembroke Welsh Corgi and the SNP site of Bichon Frise)
The SNP site of the present invention can design the chip for canine ore grade indexes, institute by being detected based on chip State the allele that chip is used in measurement table 2 give SNP site.Electedly, the chip is used in measurement table 1 give The allele of SNP site.It is further preferred that the canine strain includes Miniature Schnauzer, Ke Li, Pembroke Wales Ke Ji Dog and Bichon Frise.More preferably, the chip is Illumina chips.
Experimental example
1. the equipotential base of 230,000 SNP sites is measured with the chip (microarray) in Illumina canine 230K sites Cause.
1) blood of each dog of Miniature Schnauzer, Ke Li, Pembroke Welsh Corgi and Bichon Frise is taken to carry Take DNA sample；
2) what Genotyping was tested comprises the following steps that：
A. it is denatured the preparation of single stranded DNA：DNA sample is denatured into single-stranded with sodium hydroxide, denaturant is then neutralized, adds Enzymatic amplification reaction solution；
B. whole genome amplification：The sample of upper step is placed on whole genome amplification in 37 degree of incubators, 37 degree of reaction 20-24 Hour；
C. the genomic fragment expanded：Fragment of the amplified production digestion into hundreds of base sizes；
D. DNA is precipitated：Product after digestion adds isopropanol, and 3000g, which is centrifuged 20 minutes, precipitates DNA, drying at room temperature one Hour；
E. dissolving DNA：Add hybridization solution, 48 degree 1 hour, vortex concussion makes DNA be substantially dissolved in hybridization solution；
F. chip, DNA and chip hybridization are put：The DNA of upper step is denatured 20 minutes at 95 degree, is cooled to room temperature starting point core Piece, pays attention to avoiding cross pollution between different samples, and the chip put is placed on 16-24 hours, Bu Yaochao in 48 degree of hybrid heaters Spend 24 hours；
G. chip is cleaned：DNA of the non-hybridized or imperfect hybridization on chip is washed off, only matched completely with chip DNA can be just retained on chip；
H. Single base extension and dyeing：To hybridize to the DNA on chip Single base extension, the base of extension are carried out as template It is being combined with dyestuff for advance modified, different bases can have corresponding dye colour to determine；
I. chip, coating, fixation are cleaned：Dyestuff unnecessary on chip is washed off, fixer is added and signal is fixed；
J. chip is scanned：The chip fixed is put into the chip slot of HiScan scanners and is scanned acquisition signal, The result of scanning further can carry out interpretation of result in the software that Illumina companies provide；
3) genotypic results are analyzed.HiScan classification systems are scanned using Illumina GenomeStudio softwares Result analyzed.Clustered according to the result of dye colour during Single base extension, according to the result of cluster by material Genotype is divided into 3 classes (AA, BB, AB).
2. we have only just accurately carried out ore grade indexes with 500 site informations in above-mentioned table 1, repeat in 1 Step.
Experimental result：
Following result confirms that we are very accurate with identifications of 500 SNP to canine kind, and with all 230,000 SNP Result it is consistent.In figure below 1-4, by canine information projection to two dimensional surface, dash area represents corresponding purebred dog number for we According to the information of all purebred dogs in storehouse.Star represents sample to be tested in the position of the plane.If star is among shade, corresponding letter It is purebred to cease for the sample to be tested.Left column is the analysis only made of 500 SNP sites, and the right side is classified as 230,000 sites of full-length genome The analysis done, it is as a result completely the same, and the Given information with known dog to be measured only is consistent.
The inventors discovered that the SNP site in table 2 is enough to distinguish Miniature Schnauzer, Ke Li, Pembroke Wales Ke Ji Dog and Bichon Frise.
Although invention has been described for combined preferred embodiment, it is to be understood that protection scope of the present invention is simultaneously It is not limited to embodiment as described herein.With reference to the explanation of the invention disclosed here and practice, other implementations of the invention Example all will be readily apparent and understand for those skilled in the art.Illustrate and embodiment is to be considered only as exemplary, this hair Bright true scope and purport is defined in the claims.

Claims (7)

1. a set of SNP site for canine ore grade indexes, the SNP site for canine ore grade indexes includes giving in table 2 SNP site is gone out.
2. the SNP site for canine ore grade indexes of claim 1, the SNP site for canine ore grade indexes include SNP site is given in table 1.
3. the SNP site for canine ore grade indexes of claim 1 or 2, the canine strain includes Miniature Schnauzer, Ke Profit, Pembroke Welsh Corgi and Bichon Frise.
4. a kind of chip for canine ore grade indexes, the chip is used in measurement table 2 the equipotential base for giving SNP site Cause.
5. the chip of claim 4, the chip is used in measurement table 1 allele for giving SNP site.
6. the chip of claim 4 or 5, the canine strain includes Miniature Schnauzer, Ke Li, Pembroke Welsh Corgi And Bichon Frise.
7. any one of claim 4-6 chip, the chip is Illumina chips.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN111549144A (en) * 2020-05-07 2020-08-18 深圳有哈科技有限公司 SNP locus for cat strain identification
CN113699255A (en) * 2020-09-02 2021-11-26 北京中科昆朋生物技术有限公司 Biomarker, kit and method for identifying dogs with aggressive behavior

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111549144A (en) * 2020-05-07 2020-08-18 深圳有哈科技有限公司 SNP locus for cat strain identification
CN111549144B (en) * 2020-05-07 2023-02-14 深圳有哈科技有限公司 SNP locus for cat strain identification
CN113699255A (en) * 2020-09-02 2021-11-26 北京中科昆朋生物技术有限公司 Biomarker, kit and method for identifying dogs with aggressive behavior
CN113699255B (en) * 2020-09-02 2023-10-20 北京中科昆朋生物技术有限公司 Biomarkers, kits and methods for identifying dogs with aggression

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