CN108998539A - Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies - Google Patents
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Abstract
The invention discloses the cabezon genome SNP marker methods based on enzymes combinations Genotyping sequencing technologies, comprising the following steps: (1) cabezon extracting genome DNA: extracting cabezon genomic DNA using phenol/chloroform/isoamyl alcohol method；(2) Adapter connector designs: synthesizing a pair of of universal linker sequence, sample sequence label and one couple of PCR primers sequence；(3) cabezon genomic DNA carries out digestion with MseI and NlaIII；(4) Adapter is connected；(5) sample and PCR amplification are mixed；(6) sequence fragment and high-flux sequence are recycled；(7) data analysis exploitation SNP site.Have the beneficial effect that molecule labelling method of the present invention it is consistent, it is repeated it is high, genome molecules label can be largely developed in the case where reducing cost.
Description
Technical field
The present invention relates to cabezon genome molecules marker development technical fields, more particularly, to based on enzymes combinations gene
The cabezon genome SNP marker method of parting sequencing technologies.
Technical background
Molecular labeling be can distinguish between biological inter-species, kind in-group and in group between individual hereditary difference DNA sequence dna piece
Section.Species identification can be carried out using molecular labeling, genetic diversity is assessed, genetic variation and genetic differentiation monitors and the sections such as individual segregation is bunched
Learn research.At present using relatively broad molecular labeling mitochondrial DNA(mitochondrial DNA), microsatellite marker
(simple sequence repeat, SSR), mononucleotide polymorphic site (single nucleotide
Polymorphism, SNP) etc..With the development of high throughput sequencing technologies and bioinformatic analysis method, molecular genetics
Research requires molecular labeling that need to have the characteristics that at low cost, hereditary information amount is big, Genotyping is high-efficient.Thus SNP marker is got over
More to become important molecular labeling.For mitochondrial DNA and SSR marker, SNP marker is big with data volume, covers
The features such as degree is high, parting is high-efficient.SNP is widely distributed in genetic coding region and noncoding region, by high throughput sequencing technologies
It can be developed in the case where low cost and obtain ten hundreds of marker sites.With the development and sequencing cost of sequencing technologies
It reduces, SNP marker has become the main mark method of population genetic, Genotyping and species identification.Skill is sequenced in Genotyping
Art (genotyping-by-sequencing, GBS) is a kind of simplification genomic sequencing technique (reduced- of low cost
Representation sequencing), a large amount of SNP markers can be developed in genome range for Genotyping.Due to
It does not depend on reference to genome, the features such as process is simple, sequencing cost is low is sequenced, GBS technology is widely used to non-mode biology
The researchs such as molecular markers development, genetic map drafting, association analysis and group's genomics.Cabezon is that one kind is distributed widely in
The rock reef ovoviviparity fish in northwest Pacific sea area have important economic value and the ecological value.Point of cabezon at present
Son label is limited only to mitochondrial DNA and microsatellite marker, and the limitation of molecular labeling has seriously affected cabezon population genetic
The monitoring of diversity and genetic structure leads to divide fisheries management unit and formulate management strategy deviation occur, finally influences brown
The rational exploitation and utilization of Chang Rockfish fishery resources.For these reasons, it is necessary to which a kind of new exploitation cabezon genome SNP is provided
The analysis method in site develops a large amount of molecular labelings under the premise of reducing cost, is subsequent gene parting and genetics research
Convenience is provided.
Summary of the invention
The purpose of the present invention is to provide it is a kind of it is consistent, repeated it is high, can in the case where reducing cost it is a large amount of
Develop the cabezon genome SNP marker side based on enzymes combinations Genotyping sequencing technologies of genome molecules label
Method.
The present invention in background technique aiming at the problem that mentioning, the technical solution taken are as follows:
Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies, comprising the following steps:
Cabezon extracting genome DNA: cabezon genomic DNA is extracted using phenol/chloroform/isoamyl alcohol method；
The design of Adapter connector: a pair of of universal linker sequence, sample sequence label and one couple of PCR primers sequence are synthesized；
Cabezon genomic DNA carries out digestion with MseI and NlaIII；
Connect Adapter；
Mix sample and PCR amplification；
Recycle sequence fragment and high-flux sequence；
Data analysis exploitation SNP site.Molecule labelling method of the present invention can develop SNP molecule in cabezon genomic level
Label, the covering gene group code area SNP and noncoding region, Genotyping sequencing technologies are that a kind of simplification genome of low cost is surveyed
Sequence technology can develop a large amount of SNP markers for Genotyping in genome range, and use the gene of MseI and NlaIII
Parting sequencing technologies greatly reduce the cost of molecular markers development, and the SNP site developed can be applied to the kind of cabezon
Research, the SNP marker method and technologies such as matter stock assessment, genetic diversity and genetic structure monitoring are stablized, and repeatability is high.
Preferably, cabezon extracting genome DNA step are as follows:
(a) muscle samples removal alcohol is placed in 1.5ml centrifuge tube, 600-700 μ l digestion buffer and 18-22 μ l is added
Proteinase K Solution, concussion mix and are placed on the digested 4-6h of 50-60 DEG C of baking oven, and every 30min rocks once；
(b) after being cooled to room temperature the step a solution digested, isometric balance phenol is added in centrifuge tube, rocks mixing
To milky solution, it is centrifuged 10-15min under the conditions of 10000-15000rpm, then repeats the above steps 2-3 times；
(c) step b is obtained and isometric -20 DEG C of pre-cooling alcohol is added in centrifuged supernatant body, be vibrated to DNA and precipitate, then
After being centrifuged 5-10min under the conditions of 2-5 DEG C, 10000-15000rpm, centrifugation is 70-80% alcohol with 250-350 μ l concentration
Solution rinses twice, supernatant is abandoned after being then centrifuged 5-10min under the conditions of 10000-15000rpm, after drying in centrifuge tube
100 μ l deionized waters are added, the DNA solution of extraction is finally placed in 4 DEG C of refrigerators and saved, for use by dissolving DNA.The present invention utilizes
Phenol/chloroform/isoamyl alcohol method extracts cabezon genomic DNA, smaller to the damage of cabezon genomic DNA, is easy to obtain
The relatively better cabezon genomic DNA of integrality is obtained, while can be centrifuged after ice bath 10-20 minutes after adding isopropanol, effect
More preferably.
Preferably, digestion end and subsequent Illumina of the design of Adapter connector with MseI and NlaIII
The primer sequence that Hiseq microarray dataset requires is consistent, and the Adapter joint sequence is by Illumina microarray dataset connector sequence
Column, sample sequence label two parts composition.
Further preferably, sample sequence label design principle is as follows:
(a) sample sequence label is 6 base sequences and cannot contain continuous two identical bases；
(b) there must be at least two distinguishing bases between sample sequence label；
(c) sample label cannot be same or similar with restriction enzyme site.
Preferably, cabezon genomic DNA carries out the specific steps of digestion with MseI and NlaIII are as follows: take 0.1-1 μ g
Cabezon genomic DNA carries out digestion with MseI and NlaIII enzymes combinations, to obtain suitable mark density.Using MseI
It is widely distributed and uniform with the restriction enzyme site of NlaIII enzymes combinations enzyme, digestion, the enzyme of generation are carried out to cabezon genomic DNA
Trimscript label diversity with higher can meet screening SNP to carry out the requirement of genetic mapping.
Preferably, the specific steps of connection Adapter are as follows: the connection of Adapter connector is used with cabezon genomic DNA
MseI and NlaIII carries out digestion and carries out in same test tube, and connector is connected by ligase with DNA enzymatic simple stage property end.
Preferably, the specific steps of mixing sample and PCR amplification are as follows: the different sample DNAs of Adapter connector will be connected
Segment mixed in equal amounts, is added PCR primer sequence and DNA synzyme carries out PCR amplification.
Preferably, the specific steps of recycling sequence fragment and high-flux sequence are as follows: expanded in electrophoresis recycling step (4)
Product is sequenced using Illumina Hiseq2000 microarray dataset.High-flux sequence development approach is simple and easy, clear standard
Really, false positive is low and experimentation cost is low, and known and unknown SNP can be detected in amplification region；Sensitivity and accuracy reach close
100%。
Preferably, the specific steps of data analysis exploitation SNP site are as follows: the initial data being sequenced is according to sample mark
Label sequence carries out sorting cluster, carries out data Quality Control by sample individual and deletes Adapter joint sequence, utilizes short sequence alignment
Software BWA extracts on sequence alignment to reference genome SNP site using SAMtools software, uses VCFtools software
SNP site of the screening for population genetic variations analysis.
Compared with the prior art, the advantages of the present invention are as follows: molecule labelling method of the present invention can be in cabezon genome
SNP marker, the covering gene group code area SNP and noncoding region are developed in level, Genotyping sequencing technologies are a kind of low
The simplification genomic sequencing technique of cost can develop a large amount of SNP markers for Genotyping in genome range, and use
The Genotyping sequencing technologies of MseI and NlaIII greatly reduce the cost of molecular markers development, the SNP site developed
It can be applied to the researchs such as germ plasm resource assessment, genetic diversity and the genetic structure monitoring of cabezon, the SNP marker method
Consistent, repeatability is high.
Detailed description of the invention
Fig. 1: the cabezon genome SNP site of exploitation carries out population genetic variations analysis in present example example 2.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies, comprising the following steps:
Cabezon extracting genome DNA: cabezon genomic DNA is extracted using phenol/chloroform/isoamyl alcohol method；
The design of Adapter connector: a pair of of universal linker sequence, sample sequence label and one couple of PCR primers sequence are synthesized；
Cabezon genomic DNA carries out digestion with MseI and NlaIII；
Connect Adapter；
Mix sample and PCR amplification；
Recycle sequence fragment and high-flux sequence；
Data analysis exploitation SNP site.Molecule labelling method of the present invention can develop SNP molecule in cabezon genomic level
Label, the covering gene group code area SNP and noncoding region, Genotyping sequencing technologies are that a kind of simplification genome of low cost is surveyed
Sequence technology can develop a large amount of SNP markers for Genotyping in genome range, and use the gene of MseI and NlaIII
Parting sequencing technologies greatly reduce the cost of molecular markers development, and the SNP site developed can be applied to the kind of cabezon
Research, the SNP marker method and technologies such as matter stock assessment, genetic diversity and genetic structure monitoring are stablized, and repeatability is high.
Above-mentioned cabezon extracting genome DNA step are as follows:
(a) muscle samples removal alcohol is placed in 1.5ml centrifuge tube, 600 μ l digestion buffer and 18 μ l Proteinase Ks is added
Solution, concussion, which mixes, is placed on 50 DEG C of baking oven digested 4h, and every 30min rocks once；
(b) after being cooled to room temperature the step a solution digested, isometric balance phenol is added in centrifuge tube, rocks mixing
To milky solution, it is centrifuged 10min under the conditions of 10000rpm, then repeats the above steps 2 times；
(c) step b is obtained and isometric -20 DEG C of pre-cooling alcohol is added in centrifuged supernatant body, be vibrated to DNA and precipitate, then
After being centrifuged 5min under the conditions of 2 DEG C, 10000rpm, 250 μ l concentration of centrifugation are twice, then 70% alcoholic solution rinses
Supernatant is abandoned after being centrifuged 5min under the conditions of 10000rpm, is added 100 μ l deionized waters after drying in centrifuge tube, dissolving DNA,
The DNA solution of extraction is finally placed in 4 DEG C of refrigerators to save, for use.The present invention is extracted brown using phenol/chloroform/isoamyl alcohol method
Chang Rockfish genomic DNA, it is smaller to the damage of cabezon genomic DNA, it is easy to get the relatively better cabezon base of integrality
Because of a group DNA, while ice bath can be centrifuged after ten minutes after adding isopropanol, better effect.
The design of above-mentioned Adapter connector is sequenced with the digestion end of MseI and NlaIII and subsequent Illumina Hiseq
The primer sequence of Platform Requirements is consistent, and the Adapter joint sequence is by Illumina microarray dataset joint sequence, sample label
Sequence two parts composition.
Wherein, sample sequence label design principle is as follows:
(a) sample sequence label is 6 base sequences and cannot contain continuous two identical bases；
(b) there must be at least two distinguishing bases between sample sequence label；
(c) sample label cannot be same or similar with restriction enzyme site.
Above-mentioned cabezon genomic DNA carries out the specific steps of digestion with MseI and NlaIII are as follows: takes 0.1 μ g cabezon base
Because of a group DNA, digestion is carried out with MseI and NlaIII enzymes combinations, to obtain suitable mark density.Using MseI and NlaIII
The restriction enzyme site of enzymes combinations enzyme is widely distributed and uniform, carries out digestion, the digestion label tool of generation to cabezon genomic DNA
There is higher diversity, screening SNP can be met to carry out the requirement of genetic mapping.
The specific steps of above-mentioned connection Adapter are as follows: the connection of Adapter connector with cabezon genomic DNA with MseI and
NlaIII carries out digestion and carries out in same test tube, and connector is connected by ligase with DNA enzymatic simple stage property end.
The specific steps of above-mentioned mixing sample and PCR amplification are as follows: the different sample dna fragments of Adapter connector will be connected
Mixed in equal amounts, is added PCR primer sequence and DNA synzyme carries out PCR amplification.
The specific steps of above-mentioned recycling sequence fragment and high-flux sequence are as follows: the product expanded in electrophoresis recycling step (4),
It is sequenced using Illumina Hiseq2000 microarray dataset.High-flux sequence development approach is simple and easy, clear and accurate, false
Positive low and experimentation cost is low, and known and unknown SNP can be detected in amplification region；Sensitivity and accuracy reach nearly 100%.
The specific steps of above-mentioned data analysis exploitation SNP site are as follows: the initial data being sequenced is according to sample label sequence
Column carry out sorting cluster, carry out data Quality Control by sample individual and delete Adapter joint sequence, utilize short sequence alignment program
BWA extracts on sequence alignment to reference genome SNP site using SAMtools software, uses VCFtools software screening method
SNP site for population genetic variations analysis.
Embodiment 2:
Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies, comprising the following steps:
(1) experimental material
This laboratory sample picks up from Rushan, Zhoushan and Port of Fangcheng offshore sea waters, each 20 tail cabezon sample of group in winter in 2015
Product, amount to 60 tail samples, and sample acquisition time is close.All samples take muscle samples, be placed in 95% alcoholic solution -20 DEG C it is cold
Freeze and saves；
(2) cabezon extracting genome DNA
(a) muscle samples removal alcohol is placed in 1.5ml centrifuge tube, 650 μ l digestion buffer and 20 μ l Proteinase Ks is added
Solution, concussion, which mixes, is placed on 56 DEG C of baking oven digested 5h, and every 30min rocks once；
(b) after solution is cooled to room temperature, 650 μ l balance phenols are added in centrifuge tube, rocks and mixes to milky solution,
It is centrifuged 10min under the conditions of 10000rpm, supernatant liquid is sucked out with 1000 μ l liquid-transfering guns；
(c) 600 μ l(same volumes are added) phenol: chloroform: isoamyl alcohol (25:24:1) extracts twice again according to step (b), will
Supernatant liquid is transferred in 1.5 new ml centrifuge tubes, 1ml-20 DEG C of pre-cooling alcohol is added, gently vibration centrifugal pipe, can observe
It precipitates to DNA, after being centrifuged 10min under the conditions of 10000rpm, twice with the rinsing of 300 μ l, 75% alcoholic solution,
Supernatant is abandoned after being centrifuged 10min under the conditions of 10000rpm, is added 100 μ l deionized waters after drying in centrifuge tube, dissolving DNA,
The DNA solution of extraction is placed in 4 DEG C of refrigerators and saves for use；
(3) Adapter joint sequence
Synthesize a pair of of universal linker sequence, 60 pairs of sample sequence labels and one couple of PCR primers sequence；
(4) cabezon genomic DNA carries out digestion with MseI and NlaIII
Digestion is carried out to cabezon genomic DNA using MseI and NlaIII enzymes combinations, reaction system is 20 μ l, including 15 μ l
Deionized water, 2 μ 10 × CutSmart of l Buffer, 0.5 μ l MseI, 0.5 μ l NlaIII, 200ng sample DNA react item
Part is 37 DEG C of 2h.Reaction system is placed in 30min at 65 DEG C later and is denaturalized restriction endonuclease, inhibits its activity；
(5) digestion products are connect with Adapter connector
Adapter connector is connected after digestion, connector connection and endonuclease reaction carry out in same test tube.NEB is added
4 buffer of Buffer and each 100 μm of ol of ATP, forward and reverse connector, add after reaction 4 buffer of NEB Buffer,
The T4 ligase of ATP and 200U.Entire reaction system, which is maintained at 22 DEG C, continues 2h, and reaction system is placed in 20 at 65 DEG C later
min；
(6) sample mixing and PCR amplification
After connection product passes through Qbit quantitatively, it is mixed into PCR test tube according to sample equivalent.By 18 times (98 DEG C of repetitive cycling
30s, 65 DEG C of 30s, 72 DEG C of 30s) after obtain to sequencing library；
(7) high-flux sequence and genome SNP exploitation
It is flat in Illumina Hiseq2000 sequencing to the sequence obtained in step (6) that library and sequencing process are built according to Illumina
High-flux sequence is carried out on platform, sequencing is sequenced mode using double end 150bp, sequencing data about 70G is obtained.
Sequencing data is clustered according to sample label, respectively obtains the raw sequencing data of 60 cabezon samples.
After carrying out quality control to initial data and remove Adapter joint sequence, arrived using the BWA-mem algorithm comparison of BWA software
Cabezon obtains bam file with reference on genome sequence.Bam file is analyzed using SAMtools software, extracts SNP
Label, obtains sam file.Using VCFtools software screening method SNP marker, obtain for the analysis of cabezon Population Genetics
SNP marker.
7376 SNP markers are obtained in the present embodiment according to the above process.Use the genome SNP marker pair of above-mentioned acquisition
3 cabezon groups carry out population genetic variations analysis, as a result, it has been found that apparent clustering relationships are presented in individual in same community,
Genetic affinity is closer between individual in Display Group, and certain genetic variation and genetic differentiation is presented between different groups, as shown in Fig. 1.
Embodiment 3:
In order to improve cabezon extracting genome DNA effect, step is advanced optimized are as follows:
(2) cabezon extracting genome DNA
(a) muscle samples removal alcohol is placed in 1.5ml centrifuge tube, 650 μ l digestion buffer and 20 μ l Proteinase Ks is added
Solution, concussion, which mixes, is placed on 56 DEG C of baking oven digested 5h, and every 30min rocks once；
(b) after solution is cooled to room temperature, 650 μ l balance phenols are added in centrifuge tube, 0.43% 16 is wherein contained in balance phenol
Alkyl trimethyl ammonium bromide and 0.054% n-amyl alcohol rock and mix to milky solution, be centrifuged 10min under the conditions of 10000rpm,
Supernatant liquid is sucked out with 1000 μ l liquid-transfering guns, rocks mixing to milky solution, cetyl trimethylammonium bromide and n-amyl alcohol
It is special to exist and play gain effect with balance phenol, the cell membrane not being destroyed on the one hand can be dissolved, in combination with polysaccharide
With the substances such as protein, the amount of phenol solubilising protein is improved, so that also can effectively be denaturalized in the case where less balance phenol
Protein, and then effectively inhibit the degradation of DNAse, while can reduce meltage of the DNA in isoamyl alcohol, so that this reality
The height of example 3 improved DNA extraction method DNA yield and purity is applied, it is easy to be quick, it is low in cost, and can be in the follow-up process
It removes completely, on finally obtained DNA solution without influence；
(c) 600 μ l(same volumes are added) phenol: chloroform: isoamyl alcohol (25:24:1) extracts twice again according to step (b), will
Supernatant liquid is transferred in 1.5 new ml centrifuge tubes, 1ml-20 DEG C of pre-cooling alcohol is added, gently vibration centrifugal pipe, can observe
It precipitates to DNA, after being centrifuged 10min under the conditions of 10000rpm, twice with the rinsing of 300 μ l, 75% alcoholic solution,
Supernatant is abandoned after being centrifuged 10min under the conditions of 10000rpm, is added 100 μ l deionized waters after drying in centrifuge tube, dissolving DNA,
The DNA solution of extraction is placed in 4 DEG C of refrigerators and saves for use.
Routine operation in operation of the present invention step is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (9)
1. the cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies, it is characterised in that: packet
Include following steps:
(1) cabezon extracting genome DNA: cabezon genomic DNA is extracted using phenol/chloroform/isoamyl alcohol method；
(2) Adapter connector designs: synthesizing a pair of of universal linker sequence, sample sequence label and one couple of PCR primers sequence；
(3) cabezon genomic DNA carries out digestion with MseI and NlaIII；
(4) Adapter is connected；
(5) sample and PCR amplification are mixed；
(6) sequence fragment and high-flux sequence are recycled；
(7) data analysis exploitation SNP site.
2. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the specific steps of the cabezon extracting genome DNA are as follows:
(a) 600-700 μ l digestion buffer and 18-22 μ l Proteinase K Solution will be added in muscle samples, concussion is mixed and is placed on
4-6h is digested at 50-60 DEG C；
(b) after being cooled to room temperature the step a solution digested, isometric balance phenol is added, rocks and mixes to milky solution,
Centrifugation, then repeats the above steps 2-3 times；
(c) step b is obtained and isometric -20 DEG C of pre-cooling alcohol is added in centrifuged supernatant body, be vibrated to DNA and precipitate, be centrifuged,
Centrifugation is that 70-80% alcoholic solution rinses twice with 250-350 μ l concentration, supernatant is abandoned after centrifugation, in centrifuge tube after drying
100 μ l deionized waters of middle addition, dissolving DNA, for use.
3. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: digestion end and subsequent Illumina of the design of the Adapter connector with MseI and NlaIII
The primer sequence that Hiseq microarray dataset requires is consistent, and the Adapter joint sequence is by Illumina microarray dataset connector sequence
Column, sample sequence label two parts composition.
4. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the sample sequence label design principle is as follows:
(a) sample sequence label is 6 base sequences and cannot contain continuous two identical bases；
(b) there must be at least two distinguishing bases between sample sequence label；
(c) sample label cannot be same or similar with restriction enzyme site.
5. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the cabezon genomic DNA carries out the specific steps of digestion with MseI and NlaIII are as follows: takes
0.1-1 μ g cabezon genomic DNA, carries out digestion with MseI and NlaIII enzymes combinations, to obtain suitable mark density.
6. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the specific steps of the connection Adapter are as follows: the connection of Adapter connector and cabezon genome
DNA carries out digestion in same test tube with MseI and NlaIII and carries out, and connector is connected by ligase with DNA enzymatic simple stage property end.
7. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the specific steps of the mixing sample and PCR amplification are as follows: the not same of Adapter connector will be connected
Product DNA fragmentation mixed in equal amounts, is added PCR primer sequence and DNA synzyme carries out PCR amplification.
8. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the specific steps of the recycling sequence fragment and high-flux sequence are as follows: in electrophoresis recycling step (4)
The product of amplification is sequenced using Illumina Hiseq2000 microarray dataset.
9. the cabezon genome SNP molecule mark according to claim 1 based on enzymes combinations Genotyping sequencing technologies
Note method, it is characterised in that: the specific steps of data analysis exploitation SNP site are as follows: the initial data being sequenced according to
Sample sequence label carries out sorting cluster, carries out data Quality Control by sample individual and deletes Adapter joint sequence, utilizes short sequence
Column compare software BWA on sequence alignment to reference genome, extract SNP site using SAMtools software, use
VCFtools software screening method is used for the SNP site of population genetic variations analysis.
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Cited By (2)
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| CN110283892A (en) * | 2019-06-10 | 2019-09-27 | 浙江海洋大学 | Based on the cabezon genescreen and method for digging for simplifying genomic sequencing technique |
| CN110551806A (en) * | 2019-09-17 | 2019-12-10 | 浙江海洋大学 | Sebastiscus marmoratus SNP detection method based on high-throughput sequencing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008220198A (en) * | 2007-03-09 | 2008-09-25 | Japan Agengy For Marine-Earth Science & Technology | Scorpion fish cell line |
| CN105349675A (en) * | 2015-12-02 | 2016-02-24 | 集美大学 | Larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion |
| CN106755384A (en) * | 2017-02-15 | 2017-05-31 | 山东省海洋资源与环境研究院 | A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon |
-
2018
- 2018-08-15 CN CN201810928870.3A patent/CN108998539A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008220198A (en) * | 2007-03-09 | 2008-09-25 | Japan Agengy For Marine-Earth Science & Technology | Scorpion fish cell line |
| CN105349675A (en) * | 2015-12-02 | 2016-02-24 | 集美大学 | Larimichthys crocea genome-wide SNP and InDel molecular marker method based on double enzyme digestion |
| CN106755384A (en) * | 2017-02-15 | 2017-05-31 | 山东省海洋资源与环境研究院 | A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon |
Non-Patent Citations (7)
| Title |
|---|
| ROBERT J.ELSHIRE等: "A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species", 《PLOS ONE》 * |
| SHENGYONG XU等: "Genomic evidence for localadaptation in the ovoviviparousmarine fish Sebastiscus marmoratuswith a background of populationhomogeneity", 《SCIENTIFIC REPORTS》 * |
| 大野泰雄等: "《高分子实验学讲座》", 30 November 1965, 上海科学技术出版社 * |
| 徐怀德: "《天然产物提取工艺学》", 30 June 2008, 中国轻工业出版社 * |
| 曹墨菊等: "《植物生物技术概论》", 31 October 2014, 中国农业大学出版社 * |
| 杨玉红等: "《生命科学综合实验指导》", 31 July 2016, 东北大学出版社 * |
| 薛蕊等: "许氏平鲉（Sebastes schlegelii） EST-SSR标记开发及通用性检测", 《海洋与湖泊》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283892A (en) * | 2019-06-10 | 2019-09-27 | 浙江海洋大学 | Based on the cabezon genescreen and method for digging for simplifying genomic sequencing technique |
| CN110283892B (en) * | 2019-06-10 | 2023-03-21 | 浙江海洋大学 | Sebastes marmoratus gene screening and mining method based on simplified genome sequencing technology |
| CN110551806A (en) * | 2019-09-17 | 2019-12-10 | 浙江海洋大学 | Sebastiscus marmoratus SNP detection method based on high-throughput sequencing |
| CN110551806B (en) * | 2019-09-17 | 2023-05-02 | 浙江海洋大学 | SNP detection method of sebastes marmoratus based on high-throughput sequencing |
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