CN108103177A - High-throughput exploitation SNP site and the method for InDel in a kind of genome from pteria martensii - Google Patents
High-throughput exploitation SNP site and the method for InDel in a kind of genome from pteria martensii Download PDFInfo
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Abstract
The invention discloses high-throughput exploitation SNP site in a kind of genome from pteria martensii and the methods of InDel.This method comprises the following steps：Extract the genomic DNA of pteria martensii strain to be measured；Utilize double-stranded segment enzymatic fragmentation genomic DNA；The reparation of DNA fragmentation end, phosphorylation simultaneously add ploy (A)；DNA fragmentation jointing after repaired simultaneously screens 300~400 bp segments with paramagnetic particle method；PCR libraries are enriched with and purifying；Upper machine sequencing；Sequencing data filtering is compared, SNP and InDel detection with reference gene group.Pteria martensii genome SNP and InDel are developed using more than high throughput sequencing technologies, speed is fast, at low cost, efficient, and a large amount of SNP and InDel can be obtained by once sequencing, of far-reaching significance for the further investigation of pteria martensii genome.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind high-throughput exploitation from pteria martensii genome
The method of SNP site and InDel.
Background technology
Single nucleotide polymorphism（Single Nucleotide Polymorphism, SNP）It is long after amplified fragments
Spend polymorphism（AFLP）, microsatellite（SSR）Molecular labeling of new generation Deng after（Lander, 1996）.Due to quantity it is more,
Density is big, genetic stability is high, the advantages that being analyzed convenient for high-throughput automation detection, and SNP marker is widely used in high density
The fields such as genetic linkage maps structure, the assignment of genes gene mapping, cultivar identification, population genetic study and marker assisted selection（Liu et
Al., 2004；Mir et al., 2013；Zhang Xiao sprouts, and 2013）.Meanwhile the accuracy and reappearance of SNP marker are also superior to it
Its molecular labeling, therefore, SNP marker has broad application prospects in genetic research.
With having the characteristics that high-throughput, low cost, the sequencing new-generation sequencing technology that error rate is low, sequencing reading length is longer
With the development of bioinformatics so that high-throughput molecular markers development is possibly realized.At present, there has been no high throughputs to develop geneva pearl
The relevant report of the method for female shellfish genome SNP marker.
The content of the invention
The present invention using high throughput sequencing technologies disposably can largely obtain SNP site in pteria martensii gene and
InDel。
The purpose of the invention is to overcome the deficiencies of the prior art and provide one kind high pass from pteria martensii genome
The method of amount exploitation SNP site and InDel.
To achieve these goals, the present invention is achieved by the following technical programs：
A kind of method of large-scale development pteria martensii genome SNP marker, which is characterized in that include the following steps：
S1. the genomic DNA of pteria martensii strain to be measured is extracted；
S2. fragmentation genomic DNA；
S3. the reparation of DNA fragmentation end, phosphorylation and plus adenine tail；
S4. by the product jointing of step S3 and 300~400 bp segments of screening；
S5.PCR libraries are enriched with and purifying；
S6. upper machine sequencing；
S7. sequencing data is analyzed.
Preferably, in step S1, genomic DNA is extracted using CTAB methods.
Preferably, in step S2, double-stranded segment enzymatic fragmentation genomic DNA is used.
Preferably, in step S2, the reaction system of the fragmentation is：μ g of genomic DNA 5ng~3, Reaction
2 μ L of Buffer, 0.12 μ L of double-stranded segment enzyme add distilled water to 18 μ L systems；Reaction condition is：37 DEG C, 15h.
Preferably, the DNA fragmentation end repairs, phosphorylation and adds the reaction system of adenine tail to be：End Prep
3 μ L of Enzyme Mix, End Repair Reaction Buffer6.5 μ L, 55.5 μ L of previous step reaction product；Reaction condition
For：20 DEG C, 30 s；65 DEG C, 30 s.
Preferably, in step S4, the jointing reaction system and condition are：60 μ L of previous step reaction product,
Blunt/TA Ligase Master Mix 15μL、NEBNext Adaptor for Illumina 2.5μL、Ligation
1 μ L of Enhancer, total volume 83.5 μ L, 20 DEG C of reaction 15min；Add 3 μ L USER enzyme, 37 DEG C of reaction 15min.
Preferably, in step S4,300~400 bp segments are screened using paramagnetic particle method.
It is highly preferred that screen 300~400 bp segments using AMPure XP beads..
Preferably, in step S5, library enrichment reaction system is：In step S5, library enrichment and purification reaction system are：
23 μ L of previous step reaction product, NEBNext High Fidelity 2X PCR Master Mix μ L 25, Index
1 μ L, Universal PCR Primer of Primer, 1 μ L.
Preferably, in step S5, library enrichment and purification reaction condition are：98℃ 30s；98 DEG C of 10s, 65 DEG C of 75s,
72 DEG C of 30s, 6~15 Xun Huans；72 DEG C, 5min.
Preferably, in step S6, upper machine sequencing carries out storehouse inspection using High Sensitivity DNA Assay Kit.
Preferably, in step S6, upper machine sequencing uses Hiseq X10 PE150.
Preferably, in step S7, sequencing data analysis is data filtering, is compared with reference gene group, SNP and
InDel is analyzed.
Preferably, in step S7, sequencing data is filtered into reads of the removal containing connector, ratio containing N is more than 10%
Reads and low quality reads obtains high quality clean reads.
Preferably, in step S7, it is compared to utilize bwa softwares with reference gene group（0.7.12）Using mem algorithms
Reads after filtering is compared onto reference gene group, alignment parameters are 32-M of-k.
Preferably, in step S7, reference gene group is http://gigadb.org/dataset/100240 downloads to obtain.
Preferably, in step S7, SNP and InDel analyses use software GATK（3.4-46）'s
UnifiedGenotyper modules carry out, and filtration parameter is-Window 4 ,-filter " QD< 2.0 || FS > 60.0
|| MQ <40.0 " ,-G_filter " GQ< 20".
Compared with prior art, the present invention has the advantages that：
The present invention can be obtained largely using high throughput sequencing technologies by once sequencing from a sample
Pteria martensii genome SNP and InDel are developed using more than high throughput sequencing technologies, speed is fast, at low cost, efficiency
Height can obtain a large amount of SNP and InDel by once sequencing, of far-reaching significance for the further investigation of pteria martensii genome.
Specific embodiment
The present invention is made with reference to specification and specific embodiment and further being elaborated, the embodiment is only used
In explaining the present invention, the scope of the present invention is not intended to limit.Test method used in following embodiments is such as without special theory
It is bright, it is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained
Material.
Embodiment 1
High-throughput exploitation SNP site and the method for InDel, specifically comprise the following steps in a kind of genome from pteria martensii：
1st, DNA is extracted.
Sample to be tested genomic DNA is extracted using CTAB methods.
2nd, fragmentation genomic DNA.
Using NEBNext DNA double chain fragmentation enzymes, double-stranded DNA is cut into 50~1000bp segments.Digestion system is
Genomic DNA（The μ g of 5ng~3）, 2 μ L of Reaction Buffer, 0.12 μ L of double-stranded segment enzyme, add distilled water to 18 μ L bodies
System, mixing, 37 DEG C, 15h.
3rd, the reparation of DNA fragmentation end, phosphorylation and plus adenine tail.
The different types of end that digestion generates is repaired, while phosphorylation modification and 3 ' ends are carried out to 5 ' ends
Add A.
Reaction system is：End Prep Enzyme Mix 3μL、End Repair Reaction Buffer (10X)
6.5 μ L, 55.5 μ L of previous step reaction product.
PCR reaction conditions are：20 DEG C, 30 s；65 DEG C, 30 s.It 4 DEG C, preserves.
4th, jointing
The reaction product jointing of previous step is subjected to PCR amplification convenient for uniform template.
Reaction system and condition are：60 μ L of previous step reaction product, 15 μ L of Blunt/TA Ligase Master Mix,
2.5 μ L of NEBNext Adaptor for Illumina, 1 μ L of Ligation Enhancer, 83.5 μ L of total volume, PCR instrument 20
DEG C reaction 15min；3 μ L USER enzyme, 37 DEG C of reaction 15min are added in previous step mixed liquor.
5th, DNA fragmentation screens.
The target DNA fragment of 300~400bp is screened with AMPure XP beads magnetic beads.
6th, PCR libraries are enriched with.
Increase starting template amount is reacted by PCR, reaches the requirement of machine amount.
Reaction system is：23 μ L of previous step reaction product, NEBNext High Fidelity 2X PCR Master Mix
1 μ L, Universal PCR Primer of μ L 25, Index Primer, 1 μ L.
PCR cycle condition：98℃ 30s；98 DEG C of 10s, 65 DEG C of 75s, 72 DEG C of 30s, 6~15 Xun Huans；72 DEG C, 5min.
It 4 DEG C, preserves.
7th, upper machine sequencing.
Upper machine sequencing carries out storehouse inspection using High Sensitivity DNA Assay Kit.Afterwards, using Hiseq X10
PE150 platforms are sequenced.
8th, sequencing data is analyzed.
（1）Data filtering：To ensure the quality of data, noise data is reduced, it is big to remove the reads containing connector, ratio containing N
In 10% reads and low quality reads, high quality clean reads are obtained；
（2）It is compared with reference gene group：It utilizesbwaSoftware（0.7.12）Using mem algorithms by the reads ratios after filtering
To in reference gene group, alignment parameters are 32-M of-k；Pteria martensii reference gene group download address：http://
gigadb.org/dataset/100240。
（3）SNP and InDel analyses：Use software GATK（3.4-46）UnifiedGenotyper modules will handle
The good SNP/InDel detections for comparing file and carrying out multiple samples, the variation detected are carried out using VariantFiltration
Filtering, filtration parameter are-Window 4 ,-filter " QD< 2.0 || FS > 60.0 || MQ <40.0 " ,-G_
filter "GQ < 20"。
Embodiment 2 detects " mass-election 1 " strain and common strain geneva pearl oyster SNP site and InDel
Method according to embodiment 1 carries out SNP site and InDel analyses, first, obtains high quality clean reads（Table 1
Reads filters Information Statistics table）.Further obtain SNP site and InDel analysis data（2 all SNP/InDel statistical forms of table；
3 each sample SNP/InDel statistical forms of table）.
1 Reads of table filters Information Statistics table：
2 all SNP/InDel statistical forms of table：
3 sample SNP/InDel statistical forms of table：
The present invention obtains two strains of pteria martensii totally 9503861 SNP sites, totally 964395 InDel sites, this method
The substantial amounts of SNP site of sample and InDel can be detected simultaneously.
Claims (10)
1. high-throughput exploitation SNP site and the method for InDel in a kind of genome from pteria martensii, which is characterized in that including
Following steps：
S1. the genomic DNA of pteria martensii strain to be measured is extracted；
S2. fragmentation genomic DNA；
S3. the reparation of DNA fragmentation end, phosphorylation and plus adenine tail；
S4. by the product jointing of step S3 and 300~400 bp segments of screening；
S5. the enrichment of PCR libraries and purifying；
S6. upper machine sequencing；
S7. sequencing data is analyzed.
2. according to the method described in claim 1, it is characterized in that, in step S1, genomic DNA is extracted using CTAB methods.
3. according to the method described in claim 1, it is characterized in that, in step S2, double-stranded segment enzymatic fragmentation gene is used
Group DNA.
4. according to the method described in claim 1, it is characterized in that, in step S2, the reaction system of the fragmentation is：Gene
The μ g of group DNA 5ng~3,2 μ L of Reaction Buffer, 0.12 μ L of double-stranded segment enzyme, add distilled water to 18 μ L systems；Instead
The condition is answered to be：37 DEG C, 15h.
5. according to the method described in claim 1, it is characterized in that, in step S3, the DNA fragmentation end is repaired, phosphorylation
And add adenine tail reaction system be：End Prep Enzyme Mix 3μL、End Repair Reaction
Buffer6.5 μ L, 55.5 μ L of previous step reaction product；Reaction condition is：20 DEG C, 30 s；65 DEG C, 30 s.
6. according to the method described in claim 1, it is characterized in that, in step S4, the jointing reaction system and condition
For：60 μ L of previous step reaction product, 15 μ L of Blunt/TA Ligase Master Mix, NEBNext Adaptor for
2.5 μ L of Illumina, 1 μ L of Ligation Enhancer, total volume 83.5 μ L, 20 DEG C of reaction 15min；Add 3 μ L
USER enzyme, 37 DEG C of reaction 15min.
7. according to the method described in claim 1, it is characterized in that, in step S4,300~400 bp pieces are screened using paramagnetic particle method
Section.
8. according to the method described in claim 1, it is characterized in that, in step S5, library enrichment and purification reaction system are：On
23 μ L of single step reaction product, NEBNext High Fidelity 2X PCR Master Mix μ L 25, Index Primer
1 μ L, Universal PCR Primer, 1 μ L.
9. according to the method described in claim 1, it is characterized in that, in step S5, library enrichment and purification reaction condition are：98
℃ 30s；98 DEG C of 10s, 65 DEG C of 75s, 72 DEG C of 30s, 6~15 Xun Huans；72 DEG C, 5min.
10. according to the method described in claim 1, it is characterized in that, in step S6, upper machine sequencing uses Hiseq X10
PE150。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111489789A (en) * | 2020-04-21 | 2020-08-04 | 华中科技大学 | Method for improving mass spectrum phosphorylation modification site identification flux and accuracy |
| CN111676298A (en) * | 2020-07-09 | 2020-09-18 | 广东海洋大学 | Pinctada martensii growth trait related KSPI gene SNP molecular marker and application thereof |
| CN113403402A (en) * | 2021-07-08 | 2021-09-17 | 广东海洋大学 | Pm-SRB gene SNP molecular marker related to content of carotene in pinctada martensii shellfish and application thereof |
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| CN102127818A (en) * | 2010-12-15 | 2011-07-20 | 张康 | Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman |
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| CN102127818A (en) * | 2010-12-15 | 2011-07-20 | 张康 | Method for creating fetus DNA library by utilizing peripheral blood of pregnant woman |
Non-Patent Citations (2)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111489789A (en) * | 2020-04-21 | 2020-08-04 | 华中科技大学 | Method for improving mass spectrum phosphorylation modification site identification flux and accuracy |
| CN111489789B (en) * | 2020-04-21 | 2021-10-15 | 华中科技大学 | Method for improving mass spectrum phosphorylation modification site identification flux and accuracy |
| CN111676298A (en) * | 2020-07-09 | 2020-09-18 | 广东海洋大学 | Pinctada martensii growth trait related KSPI gene SNP molecular marker and application thereof |
| CN113403402A (en) * | 2021-07-08 | 2021-09-17 | 广东海洋大学 | Pm-SRB gene SNP molecular marker related to content of carotene in pinctada martensii shellfish and application thereof |
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Application publication date: 20180601 |