CN105349675B - Larimichthys crocea full-length genome SNP and InDel molecule labelling method based on double digestion - Google Patents
Larimichthys crocea full-length genome SNP and InDel molecule labelling method based on double digestion Download PDFInfo
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- CN105349675B CN105349675B CN201510871364.1A CN201510871364A CN105349675B CN 105349675 B CN105349675 B CN 105349675B CN 201510871364 A CN201510871364 A CN 201510871364A CN 105349675 B CN105349675 B CN 105349675B
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a kind of Larimichthys crocea full-length genome SNP and the InDel molecule labelling method based on double digestion, comprising the following steps: 1) joint sequence designs；2) genomic DNA digestion；3) joint sequence connects；4) sample mixing and PCR amplification；5) high-flux sequence；6) sequencing data analysis mining SNP site；The present invention is combined with modern molecular biology and advanced high throughput sequencing technologies, pass through the method using the combination of EcoRII and NlaIII double digestion, digestion is carried out to the DNA molecular of full-length genome, the DNA fragmentation for obtaining specific length carries out building library sequencing and SNP marker is excavated, and then obtains the equally distributed SNP site information of full-length genome；Method of the invention greatly reduces the workload and cost of full-length genome labeled analysis, is equally also suitable for other species full-length genome labeled analysis；It can be used for the research fields such as plant and animal species identification, the analysis of kind Genetic lineages, Genetic Diversity of Germplasm analysis and genetic breeding using the SNP marker that the present invention excavates.
Description
Technical field
It is specifically a kind of based on the big of double digestion the present invention relates to Larimichthys crocea full-length genome molecular markers development technical field
Yellow croaker full-length genome SNP and InDel molecule labelling method.
Background technique
Molecular labeling refer to can reflect between bion or between population in genome certain difference characteristic DNA fragmentation or alkali
Base, it directly reflects the difference between genomic DNA, thus, molecular labeling is the label on gene level, is biological heredity point
A kind of important tool of son；It mainly include RFLP (Restriction Fragment Length Polymorphism), RAPD
(Random Amplified Polymorphic DNA)、AFLP (Amplified Fragment Length
Polymorphism)、
SSR (Simple Sequence Repeat), SNP (Single Nucleotide Polymorphisms) and
InDel (small Insert and Deletion) etc.；Currently, with the development of genotyping technique, except SSR, SNP and
InDel label outside being widely used, used fewer and fewer by other kinds of label, although SSR has easy to detect, polymorphism
The features such as high, but the today being constantly progressive in high-flux sequence and analytical technology, SNP and InDel become it is more and more important
Molecular labeling, first is had the following characteristics that for SSR, widely distributed: it is wide that SNP and InDel is distributed in genome
General region, including code area and noncoding region；Second, site-specific: it is more that SNP and InDel label can explicitly point out generation
The reason of state property, but the polymorphism of SSR also needs further sequence verification；Third, high-throughput: SNP and InDel label can be with
High-throughput exploitation is carried out using the method for large scale sequencing, the number for the molecular labeling generally developed can arrive tens of thousands of to thousands of
Ten thousand；With the progress of high-flux sequence method and the further rapid decrease of cost, SNP and InDel label now at
For the main mark type of molecular labeling and genotype identification, currently, SNP and InDel label has succeeded and has widely answered
For the genetic map construction of Important Economic species, in important character QTL positioning and whole-genome association；Larimichthys crocea is one
Kind is distributed mainly on the important fish in China and East Asia, is China's yield and the maximum marine economy fish of nursery amount, rheum officinale
Research of fish important economical trait, such as the speed of growth etc. need a large amount of full-length genome SNP and InDel molecular labeling；It is existing
In technology, the exploitation of the SNP and InDel molecular labeling of Larimichthys crocea relies primarily on the side of full-length genome and transcript profile high-flux sequence
Method；Although transcript profile can be quickly obtained the site SNP and InDel in lots of genes expression area, expression area only accounts for full base
Because of 2% or so of group, the label of full genome can not be obtained；Although genome sequencing can full genome level carry out SNP and
InDel exploitation, but due to its high cost, high data analytic intensity limits it in the extensive of Larimichthys crocea SNP and InDel label
Using；For these reasons, it needs to improve the Larimichthys crocea molecule labelling method of the prior art.
Summary of the invention
Existing transcript profile and full-length genome weight sequencing technologies can be overcome in rheum officinale the purpose of the present invention is to provide a kind of
Fish molecular labeling label excavate on be unevenly distributed the problem excessively high with cost, can with lower cost within the scope of full-length genome into
The high-throughput SNP marker of row excavates Larimichthys crocea full-length genome SNP and the InDel molecule of the double digestion based on EcoRI and NlaIII
Labeling method, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme:
A kind of Larimichthys crocea full-length genome SNP and InDel molecule labelling method based on double digestion, comprising the following steps:
1) joint sequence designs；
2) genomic DNA digestion；
3) joint sequence connects；
4) sample mixing and PCR amplification；
5) high-flux sequence；
6) sequencing data analysis mining SNP site.
As a further solution of the present invention: Larimichthys crocea full-length genome SNP and the InDel molecule based on double digestion
Labeling method, specifically includes the following steps:
1) joint sequence designs: the design of joint sequence and double enzyme ends of EcoRI and NlaIII and subsequent
The primer sequence that Illumina Hiseq microarray dataset requires is consistent, and joint sequence is by microarray dataset joint sequence and sample label
Two part of sequence composition；Sample label is designed according to following principle:
It a. must a minimum of two distinguishing bases between each sample label sequence；
B. sample label sequence cannot contain continuous two identical bases；
C. sample label cannot include and cannot form restriction enzyme site distinguished sequence with joint sequence；
2) genomic DNA digestion: the genomic DNA of 200ng carries out two digestions simultaneously in the reaction system of 20 ul
Reaction；
3) joint sequence connects: joint sequence is connected in the same test tube of genomic DNA digestion and carries out, by connecing for design
Header sequence is connected on the digestion end of DNA fragmentation by ligase；
4) sample mixing builds library from PCR amplification: the DNA fragmentation of different individual of sample jointing sequences being carried out equivalent and is mixed
It closes, guarantees the harmony of each sample DNA segment number, PCR primer sequence is added and DNA synzyme carries out PCR reaction, amplification
Connection product；
5) high-flux sequence: the amplified production in step 4 is surveyed using Illumina Hiseq2000 microarray dataset
Sequence；
6) sequencing data analysis mining SNP site: sequencing initial data passes through the special sequence label of sample first and is divided
Choosing, obtains the sequencing read sequence of each sample, and the read sequence of each sample is compared using short sequence alignment program BWA
SNP excavation is carried out onto the reference genome of Larimichthys crocea, and using GATK.
Compared with prior art, the beneficial effects of the present invention are: the present invention uses the Larimichthys crocea full genome based on double digestion
Group SNP and InDel molecule labelling method can carry out SNP marker excavation in Larimichthys crocea full-length genome level, contain gene volume
Code and non-coding region；And the method due to using digestion and specific DNA fragments to recycle, greatly reduces sequence and is sequenced
Range, to efficiently control marker development cost；Present invention employs the method for EcoRI and NlaIII enzymes combinations,
DNA fragmentation containing two kinds of restriction enzyme sites in amplification Larimichthys crocea full-length genome that can be special, so that reaching simplified gene sets up library
With the purpose of sequencing；The present invention greatly reduces high throughput in Larimichthys crocea full-length genome SNP excavation and builds library, sequencing and SNP exploitation
Complexity and workload, shorten using full-length genome range SNP carry out cultivar identification, kind Genetic lineages analysis, kind
It the duty cycle of the research fields such as matter resource analysis of genetic diversity and genetic breeding, can be complete in Larimichthys crocea with lower cost
SNP excavation is carried out on genome, is Larimichthys crocea cultivar identification, the analysis of kind Genetic lineages and Genetic Diversity of Germplasm point
Analysis, the research fields such as genetic breeding provide effective molecule labelling method.
Detailed description of the invention
Fig. 1 is molecular labeling flow chart of the invention.
Fig. 2 is the fragment length distribution map for carrying out double digestion in application example of the present invention and building library.
Fig. 3 is to carry out group clustering analysis using the full-length genome SNP marker excavated in application example of the present invention.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.
Embodiment 1
Please refer to Fig. 1-3, a kind of Larimichthys crocea full-length genome SNP and InDel molecule labelling method based on double digestion, including
Following steps:
1) experimental material is chosen
This experiment takes 1 age of contemporaneity cultured large yellow croaker random population, 96 tail, and weight range and the long range of body are similar；Institute
After the characteristic index for having Larimichthys crocea individual record basic, fin ray is stayed, is saved, is put under -20 °C with the alcohol that volume fraction is 70%
It saves backup.
2) Larimichthys crocea extracting genome DNA and preservation
A. the fin of freezing is used in liquid nitrogen mortar grinder at powder, taking specification is the centrifuge tube of 1.5ml, and 1ml is added
Buffer is digested, gently powder is added in 1ml digestion buffer, soaks powder uniformly on the surface of digestion buffer,
Then shaking submerges sample, and 5h is digested at 37 DEG C, and per half an hour shakes；
B. it is cooled to room temperature to solution, is distributed into two pipes, every pipe is drawn 0.5ml balance phenol and is gently mixed, until being formed
Emulsus is centrifuged 10min under the conditions of 3000-5000 × g, removes water phase with macropore suction pipe；
C. with 0.5ml(same volume) phenol: chloroform: isoamyl alcohol (25:24:1) extracts twice again, and water phase is transferred to
In clean pipe, NaCl to concentration 0.3M(3M NaCl is added and adds 50ul), the ethyl alcohol of 2.5 times of volumes is gently injected along tube wall, gently
Jog moves pipe, until solution is thoroughly mixed, what can be will be apparent that sees that DNA rapid precipitation is got off, and is centrifuged under the conditions of 3000 × g
After 10min, twice with the rinsing of appropriate 70% ethyl alcohol, centrifugation is abandoned supernatant, is dried；
D. sample settling flux in 0.5ml TE is added NaCl to final concentration 100mM(3M NaCl and adds 16.7ul), it is added
(DNAse free) RNAse A to concentration 100ug/ml(10mg/ml RNAse A adds 5ul), after keeping 3h at 37 DEG C, add
Enter SDS to ultimate density 0.2%(10%SDS and add 10ul), and with same volume phenol: chloroform: isoamyl alcohol (25:24:1) extracts
Twice, water phase is transferred in clean pipe, NaCl to concentration 0.3M(3M NaCl is added and adds 50ul), gently injected along tube wall
The ethyl alcohol of 2.5 times of volumes, gently shakes pipe, until solution is thoroughly mixed, what can be will be apparent that is seen under DNA rapid precipitation
Come, DNA is placed on settling flux in 0.5ml 10mM TE, last sample measures OD value to determine concentration at 260nm, extracts
DNA sample be placed under -20 °C and save backup.
3) joint sequence design and synthesis
Attached drawing 2 is please referred to, the joint sequence that the present embodiment has designed and synthesized attached drawing 2 carries out high-flux sequence and builds library.
4) genomic DNA digestion
Larimichthys crocea genomic DNA (about 200ng), which is added in the reaction system of 20 ul, carries out digestion, the reaction system packet
NEB4 buffer (NEB Buffer 4), the PstI restriction endonuclease of 8U and the MspI restriction endonuclease of 8U are included, entire digestion process is at 37 °
Continue 2h under C, reaction system is placed in the activity of 65 °C of lower 20min denaturation PstI and MspI later, inhibits endonuclease reaction.
5) genomic DNA digestion products are connect with joint sequence
Joint sequence connection reaction carries out in the same test tube of genomic DNA endonuclease reaction, is equally first added No. NEB4
The reverse Y type of the corresponding forward connector and 15 pmol of 0.1 pmol is added in test tube by buffer (NEB Buffer 4) and ATP
Joint sequence；Later, the T4 ligase of NEB4 buffer, ATP and 200U are added into the reaction system of each sample, entirely
Reaction system, which is maintained under 22 °C, continues 2h, and reaction system is placed in 65 °C of lower 20min later.
6) sample mixing and amplified library
After connection product passes through Qbit accurate quantitative analysis, into a PCR test tube, mixing sample passes through mixed in equal amounts between sample
Pcr amplification reaction (95 °C, 30s of 18 wheels；62°C,30s；68 °C, 30s), equivalent amplification is carried out to mixing sample, is obtained to be measured
Preface library.
7) high-flux sequence and full-length genome SNP are developed
Amplified production in step 6 builds library and sequencing process according to Illumina and carries out high pass measurement using Hiseq platform
Sequence, sequencing use both-end 2X100bp mode, and sequencing data about 50G is obtained in average each individual sequencing 500M.
Obtained primitive sequencer read is classified according to the sequence of the special label of sample, respectively obtains 96 individual originals
Beginning read data；After deleting the special label of its sample to each read sequence, is compared using BWA to Larimichthys crocea and refer to genome
In sequence, sequence alignment bam file is obtained；Resulting bam file is analyzed using GATK, to the more of each base level
State property is judged, the SNP marker of Larimichthys crocea full-length genome is obtained.
30194 SNP polymorphism marks are obtained in the present embodiment according to the above process, wherein 12983 are 128 rheum officinales
Common to fish individual；In order to obtain this 128 individual Genetic lineages information, the full genome SNP marker base of above-mentioned acquisition is utilized
Because of type, genetic distance is carried out to this 128 fishes and has been analyzed, as a result, it has been found that have apparent cluster between some of individuals,
Show that these individuals may be the offspring of the same family, as shown in Fig. 3.
The preferred embodiment of the patent is described in detail above, but this patent is not limited to above-mentioned embodiment party
Formula within the knowledge of one of ordinary skill in the art can also be under the premise of not departing from this patent objective
It makes a variety of changes.
Claims (1)
1. a kind of Larimichthys crocea full-length genome SNP and InDel molecule labelling method based on double digestion, it is characterised in that: utilize
EcoRI and NlaIII combination carries out double digestion to genome, achievees the purpose that simplified gene sets up library and sequencing, utilizes two kinds
DNA restriction endonuclease EcoRI and NlaIII combination carry out digestion to all genes of individuals groups, obtain the DNA fragmentation of appropriate length, into
Row builds library sequencing；The requirement of digestion end and microarray dataset for two kinds of DNA restriction endonucleases EcoRI and NlaIII, designed joint
Sequence and sample label sequence；High-flux sequence, which is carried out, using the genome digestion products of EcoRI and NlaIII builds library and sequencing,
And carry out full-length genome SNP excavation；It the described method comprises the following steps:
1) joint sequence designs: double enzyme ends and subsequent Illumina of the design of joint sequence with EcoRI and NlaIII
The primer sequence that Hiseq microarray dataset requires is consistent, and joint sequence is by microarray dataset joint sequence and sample label sequence two
It is grouped as；Sample label is designed according to following principle:
A. a minimum of two distinguishing bases between each sample label sequence；
B. sample label sequence does not contain continuous two identical bases；
C. sample label does not include and does not form restriction enzyme site distinguished sequence with joint sequence；
2) genomic DNA digestion: the genomic DNA of 200ng carries out two endonuclease reactions simultaneously in the reaction system of 20 ul；
3) joint sequence connects: joint sequence is connected in the same test tube of genomic DNA digestion and carries out, by the connector sequence of design
Column are connected on the digestion end of DNA fragmentation by ligase；
4) sample mixing builds library from PCR amplification: the DNA fragmentation of different individual of sample jointing sequences is subjected to mixed in equal amounts,
Guarantee the harmony of each sample DNA segment number, PCR primer sequence is added and DNA synzyme carries out PCR reaction, amplification connects
It practices midwifery object；
5) high-flux sequence: the amplified production in step 4 is sequenced using Illumina Hiseq2000 microarray dataset；
6) sequencing data analysis mining SNP site: sequencing initial data passes through the special sequence label of sample first and is sorted, and obtains
To the sequencing read sequence of each sample, the read sequence of each sample is compared using short sequence alignment program BWA to rheum officinale
On the reference genome of fish, and SNP excavation is carried out using GATK.
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| CN106086193B (en) * | 2016-06-27 | 2019-08-06 | 山西医科大学 | A method of mixing sample DNA is analyzed based on INDEL-SNP linkage relationship |
| CN106191246B (en) * | 2016-07-12 | 2019-12-31 | 集美大学 | Method for genotyping pseudosciaena crocea genome by using liquid phase capture |
| CN107937500A (en) * | 2017-11-17 | 2018-04-20 | 深圳华大生命科学研究院 | Batch obtains the method and kit of high-precision insect COI genetic barcodes |
| CN107841544A (en) * | 2017-12-14 | 2018-03-27 | 浙江海洋大学 | A kind of method for obtaining Japanese eel high density SNP marker |
| CN108998539A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109022558A (en) * | 2018-08-15 | 2018-12-18 | 浙江海洋大学 | The flat Rockfish genome SNP marker method of Xu Shi based on enzymes combinations Genotyping sequencing technologies |
| CN109082461A (en) * | 2018-08-15 | 2018-12-25 | 浙江海洋大学 | Collichthys lucidus genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109182545B (en) * | 2018-10-18 | 2021-12-17 | 浙江海洋大学 | SNP marker associated with vibrio harveyi disease of large yellow croaker, primer and application thereof |
| CN113782103A (en) * | 2021-10-26 | 2021-12-10 | 大连大学 | DNA matrix processing method based on combined enzyme digestion mechanism |
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