CN107862177A - A kind of construction method for the SNP molecular labeling collection for distinguishing carp colony - Google Patents
A kind of construction method for the SNP molecular labeling collection for distinguishing carp colony Download PDFInfo
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Abstract
The invention discloses a kind of construction method for the SNP molecular labeling collection for distinguishing carp colony, first downloads carp weight sequencing sequence from ncbi database, is re-assemblied by composite software；The carp of download weight sequencing sequence is converted into FASTQ forms from SRA forms again, then the sequence to re-assembly is used as reference sequences, progress mapping, obtains original VCF files；The screening of molecular labeling is carried out on the basis of original VCF files；VCF files after screening compare the front and rear identification result of screening, obtain distinguishing the SNP molecular labeling collection of carp colony by the SNPRelate bags difference drawing system chadogram of R3.1.14 softwares.The SNP molecular labeling collection that the inventive method obtains can be used for the mononucleotide polymorphic site collection for differentiating multiple carp kinds, it can also be used to the selective clearing analysis based on genome high flux data.
Description
Technical field
The invention belongs to Fish genomes information extraction technology field, more particularly to a kind of mononucleotide for distinguishing carp colony
The construction method of polymorphic molecular marker collection.
Background technology
Carp is the important economic freshwater fish that China's cultured area is wide, strong stress resistance, yield are big.According to《China Fisheries count
Yearbook》, the large fresh water fish crop in China in 2011 is up to 1698.50 ten thousand tons, and wherein carp yield is 271.82 ten thousand tons.As can be seen that
The production of carp occupies very important status in China.
Understanding to worldwide carp germplasm data, is based especially on the understanding of its hereditary basis, is that we protect
With the basis using carp germplasm, even more China's development carp kind industry is basic.Germplasm Identification technology is the label of germ plasm resource,
It is control kind, the requirement of conservation.It is actually rare to be currently able to the molecular labeling of disposably 3 carp kinds of identification, more can not be to more
The carp of kind is identified.
The content of the invention
It is an object of the invention to provide a kind of monokaryon for distinguishing carp colony to overcome above the deficiencies in the prior art
The construction method of nucleotide polymorphism molecular labeling collection, find the molecular labeling that can distinguish multiple different carp kinds.
Technical scheme is as follows：
A kind of construction method for the SNP molecular labeling collection for distinguishing carp colony, comprises the following steps：
(1) carp weight sequencing sequence is downloaded from ncbi database, group again is carried out by composite software SOAP de novo2
Dress；
(2) the carp weight sequencing sequence of download is converted into FASTQ forms from SRA forms, then the sequence to re-assembly
As reference sequences, mapping is carried out by software BWA, obtains original VCF files；
(3) screening of molecular labeling is carried out on the basis of original VCF files；
(4) the VCF files after screening are compared by the SNPRelate bags difference drawing system chadogram of R3.1.14 softwares
The front and rear identification result of screening, obtain distinguishing the SNP molecular labeling collection of carp colony.
The construction method of the described SNP molecular labeling collection for distinguishing carp colony, from SRA lattice in step (2)
Formula is converted to FASTQ forms and uses fastq-dump v2.2.2 softwares.
The construction method of the described SNP molecular labeling collection for distinguishing carp colony, described in step (2)
The step of mapping, is as follows：
(1) reference gene group is built and indexed, generate the amb based on reference gene group, ann, bwt, pac, sa lattice
Several files of formula；
(2) find input and read long SA coordinates, this is the sai files for generating corresponding 2 files of pair-end sequencings；
(3) the comparison file of sam forms is generated；
(4) the sam files generated by BWA are reordered, the SAM files after sequence are then converted into bam files,
Sort sequences processing is carried out to bam files again and adds head processing；If a sample is divided into multiple by being sequenced, this step
Should be by the bam Piece file mergences of each passage；
(5) remove repetition using picard tools, remove the file after repetition and corresponding rope is formed by samtools
Quotation part, form are bai files；
(6) using the RealignerTargetCreator in GATK and IndelRealigner to causing mistake by indel
The region matched somebody with somebody is compared again, and the comparison error rate near indel is preferably minimized；
(7) using BaseRecalibrator and PrintReads by by mass value correct data output to newly
In bam files, detected for follow-up variation；Bam files are compressed using ReduceReads, generate new bam texts
Part；
VCF files are formed using samtools.
The construction method of the described SNP molecular labeling collection for distinguishing carp colony, molecule mark in step (3)
The standard of the screening of note is as follows：
1) all individual Single locus coverages must be more than 1*；
2) read length and be more than 10, mass fraction is more than 20, and minimum gene frequency is more than 0.1；
3) 1% genetic abnormality value；
4) each individual of all polymorphic sites should have corresponding base, in the absence of deletion condition.
The SNP molecular labeling collection for distinguishing carp colony that described construction method obtains is differentiating carp kind
In application.
Described application, the SNP molecular labeling collection of the differentiation carp colony can distinguish 10 Ge Li colonies,
The 10 Ge Li colonies include national kind beyond 3 China kinds and 7 China.
Described application, it is characterised in that the process for differentiating carp kind is to take the DNA of carp to be identified, is sequenced, and is sequenced
VCF files are formed by construction method described in claim 3 afterwards, and with VCF Piece file mergences in claim 3, wherein with power
SNPs positions are different in VCF files during profit requires 3, and using VCF files in claim 3 as foundation, unnecessary SNPs is removed,
Then drawing system chadogram, qualification result is obtained.
The domestic and international main culturing carps kind 10 of present invention collection, extracts their DNA and detects its quality, then carry out
High-flux sequence, obtain original lower machine data (sequencing uses Illumina HiSeq2000 pair end methods).Due to carp
Genome has been announced, therefore the present invention need not carry out de novo sequencings, and data can be downloaded from gene pool, therefore not
Need to carry out resurveying sequence, only need to download original lower machine data.
After obtaining initial data, the quality control of sequencing data is carried out by a series of indexs, ensures the accuracy of data,
Then the comparison with reference gene group is completed by instruments such as BAM, SAMtools, the extraction in mononucleotide site, forms VCF
Document.
After VCF documents are obtained, the index such as the coverage that must all is fulfilled for according to all individuals, genetic abnormality value enters line position
The screening of point, the VCF files after being screened.On this basis, the making of chadogram is completed according to SNPRelateR bags, and
Contrasted with not garbled 2 figures, analyze the result of cluster.
After it is determined that reaching the VCF files of target call, the feature of the VCF files is analyzed.This step mainly by with reference
The comment file expansion of genome.
Construction method provided by the invention obtains the SNPs data that can be used for differentiating 10 carp kinds by a series of screenings
Collection, the data set disposably can distinguish 10 carp kinds, and be divided into 4 major classes.The data set is just for dividing
10 carp kinds of analysis；The advantage of the data set can also get together the Different Individual of same kind, that is, plant cohesion one
Rise, inter-species can be made a distinction, and the result of the data set is demonstrated from clustering tree.
The development of high throughput sequencing technologies in recent years and the reduction of sequencing cost cause from all molecular labelings of genomic DNA
It is possibly realized to carry out Germplasm Identification, also for multiple carp kinds identify providing approach simultaneously, and can fundamentally obtain
Obtain these molecular labeling collection that can be used for multiple carp kinds to distinguish.The present invention passes through both at home and abroad totally 10 based on carp weight sequencing technologies
Individual carp kind, 30 fishes, have found a kind of SNP molecular labeling collection for distinguishing 10 Ge Li colonies altogether.
The present invention screens from all polymorphic site data sets based on genome can distinguish the more of 10 kinds of carp
State site, it is however generally that, the polymorphic sites of 2~3 different cultivars can be distinguished by, which being filtered out by functional gene, has been not easy,
If 10 kinds are just more difficult to；The polymorphic site that the present invention is obtained by high flux data screening just has 38796, this
It is that traditional candidate gene microsatellite or both combinations can not be accomplished.
Can be successfully in dendrogram by 10 with the SNP molecular labeling collection of method provided by the invention
Individual carp cultivar identification comes, and the Different Individual of same kind can get together, and not intersect the situation of cluster, by building
Erection system chadogram can verify this result, but Heilongjiang Carp Different Individual can not get together, therefore resolution
For more than 90%.This molecular labeling collection is applied to the control kind of carp, can promote the development of carp kind industry and the protection of property right, while
It is assurance and understands the basic and basic of global carp germ plasm resource.
Brief description of the drawings
Fig. 1 is the dendrogram that 10 carp kind NJ clustering procedures are drawn in the embodiment of the present invention 1, specially 10 carp kinds 30
Individual Clustering Effect figure；
Fig. 2 is the dendrogram drawn in the embodiment of the present invention 1 to 3 carp individuals using the data set, has specially been screened
Species estimation design sketch of the SNP data sets to 3 unknown individuals.
Embodiment
Embodiment 1
I) obtain the mononucleotide polymorphic site collection for differentiating 10 different cultivars carps
30 weight sequencing sequences (PRJNA202478) of carp are downloaded from NCBI (www.ncbi.nlm.nih.gov) database
(the individual source of this 30 sequences and the visible following article of sequence measurement：Xu P,Zhang XF,Wang XM,et al.
Genome sequence and genetic diversity of the common carp,Cyprinus
carpio.Nature genetic, 2014,46(11):1212-1219.), specific Latin literary fame, Chinese name and English contracting
Write and be shown in Table 1, altogether 10 kinds, the repeat number of each kind is 3~4.Composite software uses SOAP de novo2, carries out weight
New assembling, assembling the results are shown in Table 2.From table 2 it can be seen that ScaffoldN50 and ScaffoldN90 are all higher than the numerical value of original text,
Prove that assembling sequence is reliable, sequencing depth is that 4~9, contig is 9377.Needed and original text after completing the assembling of genome
Assembling result compare, Scaffold N50 value must not be less than data value during former paper publishing.
Fastq-dump v2.2.2 softwares are downloaded from NCBI, are changed the sequence of download from SRA forms by the software
For FASTQ forms, then using the sequence of above-mentioned assembling as reference sequences, mapping is carried out by software BWA:
1) reference gene group is built and indexed, generate the amb based on reference gene group, ann, bwt, pac, sa forms
Several files；
2) find input and read long SA coordinates, this is the sai files for generating corresponding 2 files of pair-end sequencings；
3) the comparison file of sam forms is generated；
4) the sam files generated by BWA are reordered, the SAM files after sequence are then converted into bam files,
Sort sequences processing is carried out to bam files again and adds head processing；If a sample is divided into multiple by being sequenced, this step
Should be by the bam Piece file mergences of each passage；
5) remove repetition using picard tools, remove the file after repetition and corresponding index is formed by samtools
File, form are bai files；
6) using the RealignerTargetCreator in GATK and IndelRealigner to causing mispairing by indel
Region compared again, the comparison error rate near indel is preferably minimized；
7) using BaseRecalibrator and PrintReads by by the data output that mass value corrects to new bam
In file, detected for follow-up variation；Bam files are compressed using ReduceReads, generate new bam files；
8) VCF files are formed using samtools.
After original VCF files are obtained, the screening of molecular labeling is carried out by standard once：
1) all individual Single locus coverages must be more than 1*；
2) read length and be more than 10, mass fraction is more than 20, and minimum gene frequency is more than 0.1；
3) 1% genetic abnormality value；
4) each individual of all polymorphic sites should have corresponding base, in the absence of deletion condition；
After garbled VCF files are obtained, by the SNPRelate bags of R3.1.14 softwares, drawing system is evolved respectively
Set (Fig. 1), compare the front and rear Germplasm Identification effect of screening.By it was found that, the mononucleotide polymorphic position obtained by screening
10 carp kinds can be divided into 4 major classes by point data collection：U.S. carp, fancy carp, Sol irrigate assorted carp, Danube carp and pine
Pu mirror carp gathers to be gathered for the 4th class for the 3rd class, the Yellow River carp, Heilongjiang Carp, Oujiang Color Common Carp, Cyprinus carpio var. color, Singuo Red Carp and C carpiovarwuyuanensis.Put
Big cluster graph discovery：Heilongjiang Carp is compared with other kinds, and more scattered, Oujiang Color Common Carp, Cyprinus carpio var. color and C carpiovarwuyuanensis are difficult to differentiate between out
Come, that is to say, that the two genetic connection is near.By the structure of systematic evolution tree it can be seen that the Different Individual of single kind is basic
On gather on same node, in the absence of intersect cluster phenomenon, from this point on from the point of view of, spe cies identification rate be 100%.
II) respectively randomly select three from 3 carp kinds, identification whether the kind of the carp
It is each from C carpiovarwuyuanensis, Songpu mirror carp and the Yellow River carp colony to select a carp at random, tail fin is cut, is distinguished after extracting DNA
Numbering is R1, R2 and R3, is sent to Nuo Hezhiyuan biological informations Co., Ltd and carries out resurveying sequence, and it is Hi- to resurvey sequence using platform
Seq × 10, sequencing depth be 10 ×, the reads obtained after sequencing, by I) described in data handling procedure, then with I)
The VCF Piece file mergences of middle acquisition, have and I) SNPs positions are different in VCF files, with I) gone for foundation, unnecessary SNPs
Fall, then do systematic evolution tree again, see Fig. 2.As a result find that 3 detected carps can be accredited out exactly.
The essential information of the carp kind of table 1. 10
2. 30 carp full-length genome assembling information of table
Claims (7)
1. a kind of construction method for the SNP molecular labeling collection for distinguishing carp colony, it is characterised in that including following
Step：
（1）Carp weight sequencing sequence is downloaded from ncbi database, is re-assemblied by composite software SOAP de novo2；
（2）The carp of download weight sequencing sequence is converted into FASTQ forms from SRA forms, then using the sequence that re-assemblies as
Reference sequences, mapping is carried out by software BWA, obtains original VCF files；
（3）The screening of molecular labeling is carried out on the basis of original VCF files；
（4）VCF files after screening compare screening by the SNPRelate bags difference drawing system chadogram of R3.1.14 softwares
Front and rear identification result, obtain distinguishing the SNP molecular labeling collection of carp colony.
2. the construction method of the SNP molecular labeling collection according to claim 1 for distinguishing carp colony, it is special
Sign is, step（2）In be converted to FASTQ forms from SRA forms and use fastq-dump v2.2.2 softwares.
3. the construction method of the SNP molecular labeling collection according to claim 1 for distinguishing carp colony, it is special
Sign is, step（2）Described in mapping the step of it is as follows：
（1）Reference gene group is built and indexed, generates the amb based on reference gene group, ann, bwt, pac, sa forms
Several files；
（2）Find input and read long SA coordinates, this is the sai files for generating corresponding 2 files of pair-end sequencings；
（3）Generate the comparison file of sam forms；
（4）The sam files generated by BWA are reordered, the SAM files after sequence are then converted into bam files, then it is right
Bam files carry out sort sequences processing and add head processing；If a sample is divided into multiple by being sequenced, this step should will be every
The bam Piece file mergences of individual passage；
（5）Remove repetition using picard tools, remove the file after repetition and corresponding index text is formed by samtools
Part, form are bai files；
（6）Using the RealignerTargetCreator in GATK and IndelRealigner to causing mispairing by indel
Region is compared again, and the comparison error rate near indel is preferably minimized；
（7）Using BaseRecalibrator and PrintReads by by the data output that mass value corrects to new bam texts
In part, detected for follow-up variation；Bam files are compressed using ReduceReads, generate new bam files；
VCF files are formed using samtools.
4. the construction method of the SNP molecular labeling collection according to claim 1 for distinguishing carp colony, it is special
Sign is, step（3）The standard of the screening of middle molecular labeling is as follows：
1）All individual Single locus coverages must be more than 1*；
2）Read length and be more than 10, mass fraction is more than 20, and minimum gene frequency is more than 0.1；
3）1% genetic abnormality value；
4）The each individual of all polymorphic sites should have corresponding base, in the absence of deletion condition.
5. the SNP molecular labeling collection for the differentiation carp colony that the construction method described in claim 1 obtains is differentiating
Application in carp kind.
6. application according to claim 5, it is characterised in that the SNP molecular labeling of the differentiation carp colony
Collection can distinguish 10 Ge Li colonies, and the 10 Ge Li colonies include national kind beyond 3 China kinds and 7 China.
7. application according to claim 5, it is characterised in that the process for differentiating carp kind is to take the DNA of carp to be identified, is entered
Row sequencing, forms VCF files, and close with VCF files in claim 3 after sequencing by the construction method described in claim 3
And wherein different from SNPs positions in VCF files in claim 3, it is unnecessary as foundation using VCF files in claim 3
SNPs is removed, and then drawing system chadogram, obtains qualification result.
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| CN110349625A (en) * | 2019-07-23 | 2019-10-18 | 中国科学院心理研究所 | A kind of method for building up of human brain gene expression space-time norm |
| CN113284552A (en) * | 2021-06-11 | 2021-08-20 | 中山大学 | Method and device for screening micro haplotype |
| CN117210580A (en) * | 2023-10-10 | 2023-12-12 | 中国水产科学研究院 | Application of SNP molecular marker combination in identification of 16 carp varieties |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113284552A (en) * | 2021-06-11 | 2021-08-20 | 中山大学 | Method and device for screening micro haplotype |
| CN113284552B (en) * | 2021-06-11 | 2023-10-03 | 中山大学 | Screening method and device for micro haplotypes |
| CN117210580A (en) * | 2023-10-10 | 2023-12-12 | 中国水产科学研究院 | Application of SNP molecular marker combination in identification of 16 carp varieties |
| CN117210580B (en) * | 2023-10-10 | 2024-02-27 | 中国水产科学研究院 | Application of SNP molecular marker combination in identification of 16 carp varieties |
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