CN109402285A - A kind of classifying method based on complete genome DNA methylation sites genotype - Google Patents
A kind of classifying method based on complete genome DNA methylation sites genotype Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The present invention provides a kind of classifying methods based on complete genome DNA methylation sites genotype, are related to plant molecular breeding technical field, the genomic DNA including extracting identical tissue sample；The DNA library of bisulfite processing is established, then is sequenced, the DNA methylation site of each sample is identified according to sequencing result, calculates methylation supporting rate, genotyping is carried out according to the DNA methylation supporting rate of methylation sites.Method provided by the invention can be realized DNA methylation site allelotype parting, and classifying method provided by the invention is to take the same tissue of Different Individual in group in same time point, can exclude the influence of environmental effect and growth conditions to methylation state of DNA.Method provided by the invention utilizes high throughput sequencing technologies method, carries out accurate parting to the genotype in DNA methylation site.
Description
Technical field
The present invention relates to plant molecular breeding technical fields, and in particular to one kind is based on complete genome DNA methylation sites
The classifying method of genotype.
Background technique
DNA methylation (DNA methylation) is one of the apparent modified types of gene found earliest, is a kind of DNA
Chemical modification type in level can cause gene function that heritable variation occurs under the premise of not changing DNA sequence dna, and
Eventually lead to the genetic mechanism of phenotypic variation.In higher eucaryote, the cytimidine (C) in DNA often occurs for DNA methylation
On, it is under the action of DNA methyltransferase, the methyl on S adenosine formyl methionine (SAM) molecule to be transferred to DNA points
In son on 5 carbon atoms of cytosine residues.And DNA methylation site can be hereditary with the duplication of DNA, because of DNA replication dna
Afterwards, methylase can methylate newly synthesized unmethylated site.
Full-length genome bisulfite sequencing (whole genome bisulfite sequencing) is that present research is complete
The most important experimental technique of genomic DNA methylation level.DNA unmethylated C base transition after bisulfite is handled is U alkali
Base (being finally T base after PCR amplification), and the C base to methylate then remains unchanged, and has methylation modification with script with this
Cytimidine distinguish, then to PCR product carry out high-flux sequence, the complete genome DNA first of single base resolution ratio can be drawn
Base map.
Covalent modification of the DNA methylation as Matrix attachment region can be realized precisely during mitosis and meiosis
Ground heredity, it is similar to the SNP that DNA sequence dna natural mutation generates, since the fidelity of dnmt rna in genome is lower,
Mistake can occur during methylation sites state-maintenance in gene, lead to the appearance of monomethylation diversity point.To these
The site SMPs carries out genotyping, can deeply be probed into the function further to DNA methylation site.
Currently, being based on full-length genome bisulfite sequencing approach, monomethylation diversity point is only obtained, without
Suitable method carries out genotyping to the allelotype in DNA methylation site.
Summary of the invention
The purpose of the present invention is to provide a kind of classifying methods based on complete genome DNA methylation sites genotype, adopt
With method provided by the invention, parting can be carried out to the allelotype in DNA methylation site.
The present invention provides a kind of classifying methods based on complete genome DNA methylation sites genotype, which is characterized in that
The following steps are included:
1) genomic DNA of the identical tissue sample of each individual in group is extracted respectively；
2) genomic DNA of each sample extracted according to the step 1) establishes the DNA library of bisulfite processing；
3) DNA library for the bisulfite processing established respectively to each sample is sequenced, and obtains each sample
Sequencing result；
4) the DNA methylation site of each sample is identified according to the sequencing result of each sample of step 3)；
5) the methylation supporting rate for calculating each DNA methylation site of each sample, according to the DNA first of methylation sites
Base supporting rate carries out genotyping；
When DNA methylation supporting rate is greater than 0.7, genotype M:M；
When DNA methylation supporting rate be [0.3~0.7) when, genotype be U:M or M:U；
When DNA methylation supporting rate is less than 0.3, genotype U:U.
Preferably, the step 2) genomic DNA establishes the method for DNA library of bisulfite processing and includes:
The genomic DNA is interrupted into the segment for 200~300bp at random；
Obtained segment is successively carried out to end modified plus A tail and connection sequence measuring joints, the segment after being connected；
Segment after the connection is subjected to bisulfite processing, the segment that obtains that treated；
Treated by described in, and segment carries out PCR amplification, obtains DNA library.
Preferably, the cytimidine of the sequence measuring joints is modified through methylation.
Preferably, the identical tissue sample of the step 1) is that Different Individual obtains at same time point under identical growing environment
It obtains.
Preferably, the depth of the step 3) sequencing be 30 ×.
Preferably, the method for the step step 4) identification includes:
T is converted as C respectively with sequencing read by the sequence that sequencing obtains and G is converted into A；
Sequencing read after sequence and conversion after conversion is compared；
DNA methylation site is identified according to optimal comparison result.
Preferably, the step 5) calculating includes: to detect to each DNA methylation site, obtains the branch in the site
It holds the read number of methylation and supports the read number of non-methylation, to calculate methylation supporting rate.
Preferably, the formula of the calculating are as follows: support methylation read number/(support methylation read number+
Hold the read number of non-methylation).
The present invention provides a kind of classifying methods based on complete genome DNA methylation sites genotype, using the present invention
The method of offer can carry out accurate parting to DNA methylation site allelotype, and equipotential DNA methylation site is in genome
In there are three types of genotype: homozygous methylation sites (M:M), i.e. the cytimidine site are in methylation shape on loci
State；Hemimethylation state (U:M or M:U), i.e. the cytimidine site are in methylation state on an allelosomal, another
Non- methylation state is on a allelosomal；Homozygous non-methylation sites (U:U), i.e., the cytimidine site is in loci
On be in non-methylation state.Theoretically, the supporting rate in the DNA methylation site of three kinds of genotype is respectively 100%, 50%
With 0%, according to statistical algorithms, it is believed that when DNA methylation supporting rate is greater than 0.7, genotype M:M；When DNA methylation branch
Holdup be [0.3~0.7) when, genotype be U:M or M:U；When DNA methylation supporting rate is less than 0.3, genotype U:U.
The embodiment of the present invention is as the result is shown: method provided by the invention can be realized DNA methylation site allele
Type parting, and classifying method provided by the invention is to take the same tissue of Different Individual in group in same time point, it can
To exclude the influence of environmental effect and growth conditions to methylation state of DNA.Method provided by the invention is measured using high pass
Sequence technical method carries out accurate parting to the genotype in DNA methylation site.
Detailed description of the invention
Fig. 1 provides DNA methylation genotyping method flow diagram by the embodiment of the present invention 1；
Fig. 2 is the DNA methylation genotyping result schematic diagram of the embodiment of the present invention 1.
Specific embodiment
The present invention provides a kind of classifying methods based on complete genome DNA methylation sites genotype, which is characterized in that
The following steps are included:
1) genomic DNA of the identical tissue sample of each individual in group is extracted respectively；
2) genomic DNA of each sample extracted according to the step 1) establishes the DNA library of bisulfite processing；
3) DNA library for the bisulfite processing established respectively to each sample is sequenced, and obtains each sample
Sequencing result；
4) the DNA methylation site of each sample is identified according to the sequencing result of each sample of step 3)；
5) the methylation supporting rate for calculating each DNA methylation site of each sample, according to the DNA first of methylation sites
Base supporting rate carries out genotyping；
When DNA methylation supporting rate is greater than 0.7, genotype M:M；
When DNA methylation supporting rate be [0.3~0.7) when, genotype be U:M or M:U；
When DNA methylation supporting rate is less than 0.3, genotype U:U.
The genomic DNA of the identical tissue sample of each individual in group is extracted respectively
The present invention extracts the genomic DNA of the identical tissue sample of each individual in group respectively.The present invention is to the sample
The extracting method of product genomic DNA is not particularly limited, using the method for traditional extraction plant genome DNA.In this hair
It is specifically extracted using plant genome DNA extracts kit in bright embodiment, DNeasy Plant Mini specifically can be used
Kit (Qiagen China, Shanghai, China) is extracted.
In the present invention, the identical tissue sample preferably under identical growing environment Different Individual at same time point
It acquires.The environmental effect of influence so as to avoid to(for) DNA methylation loci gene type parting.
The present invention establishes the DNA library of bisulfite processing according to the genomic DNA of each sample of extraction.
In the present invention, the method that the genomic DNA establishes the DNA library of bisulfite processing preferably includes: will
The segment that the genomic DNA is interrupted at random as 200~300bp；Obtained segment is successively carried out it is end modified, plus A tail and
Connect sequence measuring joints, the segment after being connected；Segment after the connection is subjected to bisulfite processing, after obtaining processing
Segment；Treated by described in, and segment carries out PCR amplification, obtains DNA library.
In the present invention, the purpose that the segment successively carries out end modified plus A tail and connects sequence measuring joints is to be rear
The continuous primer required for sequencing procedure in synthesis provides sequence information.The end modified plus A that the present invention carries out the segment
The method of tail and connection sequence measuring joints is not particularly limited, using routine.In the present invention, the born of the same parents of the sequence measuring joints are phonetic
Pyridine is preferably modified through methylation.The present invention is not particularly limited the method for the segment after bisulfite processing connection,
Using conventional method, after treatment with heavy sulfites after, the C not methylated becomes U (becoming T after PCR amplification), and
The C of methylation is remained unchanged.The method that the present invention carries out PCR amplification to treated the segment is not particularly limited, and is used
Conventional method.
The DNA library for the bisulfite processing that the present invention respectively establishes each sample is sequenced, and obtains each sample
The sequencing result of product.In the present invention, the depth of the sequencing be preferably 30 ×.The present invention is to the no special limit of sequencing
It is fixed, using conventional sequencing approach.
The present invention identifies the DNA methylation site of each sample according to the sequencing result of each sample.In the present invention, institute
The method for stating identification preferably includes: converting T as C respectively with sequencing read for the sequence that sequencing obtains and G is converted into A；It will turn
The sequencing read after sequence and conversion after change is compared；DNA methylation site is identified according to optimal comparison result.
Present invention preferably employs the reference genomes that Bismark software (bottom calls Bowtie2) carries out methylation data
Comparison analysis.
The present invention calculates the methylation supporting rate in each DNA methylation site of each sample, according to methylation sites
DNA methylation supporting rate carries out genotyping；
When DNA methylation supporting rate is greater than 0.7, genotype M:M；
When DNA methylation supporting rate be [0.3~0.7) when, genotype be U:M or M:U；
When DNA methylation supporting rate is less than 0.3, genotype U:U.
In the present invention, the calculating preferably includes: detecting to each DNA methylation site, obtains the site
It supports the read number of methylation and supports the read number of non-methylation, to calculate methylation supporting rate.
In the present invention, the formula of the calculating is preferred are as follows: support methylation read number/(support methylation
Read number+support non-methylation read number).
In the present invention, the group preferably includes Chinese white poplar natural population.
Combined with specific embodiments below to of the present invention a kind of based on complete genome DNA methylation sites genotype
Classifying method is further described in detail, and technical solution of the present invention includes but is not limited to following embodiment.
Embodiment 1
The acquisition of sample: 300 plants of individuals are randomly selected in Chinese white poplar germplasm resource bank；
Plant genome DNA extracts kit is commercial product；
The identification in DNA methylation site is carried out using Bismark software (bottom calls Bowtie2).
Specific steps are as follows:
Step S1 randomly selects 300 plants of individuals as survey from plantation in the Chinese white poplar germplasm resource bank of Shandong Province Guan County
Sequence group, Yu Shangwu 9:00~11:00 acquire its functional leaf (climax leaves of branch upper end the 3rd~5), in order to prevent its DNA first
Base state changes, and is immediately placed in liquid nitrogen environment (- 196 DEG C) and saves after having acquired.
Step S2 utilizes DNeasy Plant Mini Kit (Qiagen China, Shanghai, China) kit pair
The genomic DNA of leaf sample extracts.
After completing above-mentioned steps, further obtained genomic DNA can be detected, S21 is with agarose gel electrophoresis
Judge the palliating degradation degree of DNA sample and whether has RNA pollution；S22 is used using RNsae-free water as blank control
Nanodrop detects the OD260/OD280 ratio of each DNA sample, determines the purity of DNA sample；S23 is using Qubit to each DNA
The concentration of sample carries out accurate quantification.
Then implementation steps S3 establishes the DNA text of bisulfite processing according to the genomic DNA of each sample of extraction
Library, the specific method is as follows:
Step S31 is interrupted genomic DNA at random to 200-300bp using Covaris S220；
Step S32, the DNA fragmentation having no progeny of fighting each other carry out end reparation plus A tail, and connect upper all cytimidines and pass through
Methylate the sequence measuring joints modified, the DNA fragmentation after being connected, and its object is to be the following sequencing procedure institute in synthesis
The primer needed provides sequence information；
DNA fragmentation after connecting in step S32 is carried out bisulfite processing, by processing, not occurred by step S33
The C of methylation becomes U (becoming T after PCR amplification), and the C to methylate is remained unchanged, and EZ DNA specifically can be used
Methylation Gold Kit (Zymo Research, MurphyAve.Irvine, CA, U.S.A.)；
Step S34 carries out PCR amplification through bisulfite treated DNA fragmentation in step S33, obtains weight sulfurous
The DNA library of hydrochlorate processing.
Implementation steps S4 send the DNA library of bisulfite processing limited to Beijing source Nuo Hezhi biological information science and technology
Company is sequenced.
Implementation steps S5, according to sequencing result identification of dna methylation sites, using Bismark software, (bottom is called
Bowtie2), the comparison analysis for carrying out the reference genome of methylation data, to identify DNA methylation site.
Implementation steps S6 calculates the methylation supporting rate in each DNA methylation site.It is specific as follows: to each DNA methyl
Change site to be detected, obtain the read number of the support methylation in the site and supports the read number of non-methylation, from
And calculate the DNA methylation supporting rate in the site, calculation formula are as follows: support methylation read number/(support methylation
Read number+support non-methylation read number).Fig. 2 shows the supports of the DNA methylation in genome portion cytimidine site
Rate frequency distribution, wherein DNA methylation supporting rate is distributed in each frequency range.
Implementation steps S7 carries out genotyping according to site DNA methylation supporting rate, if DNA methylation supporting rate >
0.7, it is M:M；DNA methylation supporting rate is then U:M or M:U between 0.3~0.7；DNA methylation supporting rate < 0.3, then for
U:U.In the present embodiment, it is exemplified by Table 1, showing is that same position Chr01_1292243 cytimidine site exists in genome
DNA methylation supporting rate in intragroup Different Individual may be implemented according to supporting rate to DNA methylation site equipotential base
Because type is in intragroup benchmark genotyping.
The genotype in 1 site Chr01_1292243 of table
Can be seen that method provided by the invention by the above experimental data can be realized DNA methylation site equipotential base
Because of type parting, and classifying method provided by the invention is to take the same tissue of Different Individual in group in same time point,
It can exclude the influence of environmental effect and growth conditions to methylation state of DNA.Method provided by the invention utilizes high throughput
Sequencing technologies method carries out accurate parting to the genotype in DNA methylation site.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of classifying method based on complete genome DNA methylation sites genotype, which comprises the following steps:
1) genomic DNA of the identical tissue sample of each individual in group is extracted respectively；
2) genomic DNA of each sample extracted according to the step 1) establishes the DNA library of bisulfite processing；
3) DNA library for the bisulfite processing established respectively to each sample is sequenced, and obtains the sequencing of each sample
As a result；
4) the DNA methylation site of each sample is identified according to the sequencing result of each sample of step 3)；
5) the methylation supporting rate for calculating each DNA methylation site of each sample, according to the DNA methylation of methylation sites
Supporting rate carries out genotyping；
When DNA methylation supporting rate is greater than 0.7, genotype M:M；
When DNA methylation supporting rate be [0.3~0.7) when, genotype be U:M or M:U；
When DNA methylation supporting rate is less than 0.3, genotype U:U.
2. classifying method according to claim 1, which is characterized in that the step 2) genomic DNA establishes weight sulfurous acid
The method of the DNA library of salt treatment includes:
The genomic DNA is interrupted into the segment for 200~300bp at random；
Obtained segment is successively carried out to end modified plus A tail and connection sequence measuring joints, the segment after being connected；
Segment after the connection is subjected to bisulfite processing, the segment that obtains that treated；
Treated by described in, and segment carries out PCR amplification, obtains DNA library.
3. classifying method according to claim 2, which is characterized in that the cytimidine of the sequence measuring joints is repaired through methylation
Decorations.
4. classifying method according to claim 1, which is characterized in that the identical tissue sample of the step 1) is in identical life
Different Individual acquires at same time point under long environment.
5. classifying method according to claim 1, which is characterized in that the depth of the step 3) sequencing is 30 ×.
6. classifying method according to claim 1, which is characterized in that the method for the step step 4) identification includes:
T is converted as C respectively with sequencing read by the sequence that sequencing obtains and G is converted into A；
Sequencing read after sequence and conversion after conversion is compared；
DNA methylation site is identified according to optimal comparison result.
7. classifying method according to claim 1 or 6, which is characterized in that the step 5) calculating includes: to each DNA
Methylation sites are detected, and are obtained the read number of the support methylation in the site and are supported the read number of non-methylation,
To calculate methylation supporting rate.
8. classifying method according to claim 7, which is characterized in that the formula of the calculating are as follows: support methylation
Read number/(the read number+support non-methylation read number for supporting methylation).
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