CN104268441B - Method for obtaining double DNA restriction endonuclease combinations based on bioinformatics - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 108091008146 restriction endonucleases Proteins 0.000 title claims abstract description 20
- 230000007023 DNA restriction-modification system Effects 0.000 title abstract 2
- 239000012634 fragment Substances 0.000 claims abstract description 96
- 230000029087 digestion Effects 0.000 claims abstract description 63
- 238000012163 sequencing technique Methods 0.000 claims abstract description 17
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims description 80
- 108090000790 Enzymes Proteins 0.000 claims description 80
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 claims description 35
- 108020004414 DNA Proteins 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 108091029795 Intergenic region Proteins 0.000 claims description 11
- 238000004088 simulation Methods 0.000 claims description 11
- 238000005516 engineering process Methods 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 238000000205 computational method Methods 0.000 claims description 3
- 210000000349 chromosome Anatomy 0.000 claims description 2
- 239000004744 fabric Substances 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 abstract 1
- 238000001712 DNA sequencing Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150036080 at gene Proteins 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
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Abstract
The invention relates to bioinformatics, in particular to a method for obtaining double DNA restriction endonuclease combinations based on the bioinformatics. The method specifically comprises the steps of designing a double-enzyme combination scheme according to restriction enzyme cutting sites of restriction endonuclease to perform simulated digestion on a target species reference genome; performing distribution analysis on the fragments ranging from 90bp to 750 bp produced in the simulated digestion; performing distribution situation statistics of the target species reference genome according to the analysis, and obtaining distribution values of different double-enzyme combinations, wherein a high-value double-enzyme combination is an optical double-enzyme combination. The method has the advantages that different double-enzyme combinations can be selected for achieving different experimental purposes, different simplifying efficiencies can be achieved, balance between experimental cost and sequencing data amount is facilitated, and accordingly an experimental scheme is optimized.
Description
Technical field
The present invention relates to bioinformatics, specifically a kind of to obtain double DNA restricted enzyme based on bioinformatics
The method of combination.
Background technology
As the development of second filial generation DNA sequencing technology, the quality of DNA sequencing result are improved constantly, sequencing cost gradually drops
It is low.Many researchers obtain the sequence information of species to be studied all using secondary sequencing technologies.For to be studied
For the project of body number less (being less than 100), there is genome sequencing sequence information to enrich, genomic coverage is wide
General advantage, but for the project of individual amount to be studied more (be more than 100), full-length genome is resurveyed the cost of sequence
Still it is very big.How to reduce sequencing cost on the basis of ensureing to obtain enough sequence informations just becomes extremely important.
At present the method for main flow is the method that RAD, GBS etc. simplify genome, and both approaches simplify the major way of genomic information
It is exactly, by DNA digestion with restriction enzyme genomic DNAs, simplified mesh to be reached by fragment of the enrichment with restriction enzyme site
's.The most important step of this mode is exactly to select suitable restricted enzyme, and suitable restricted enzyme should meet
Several conditions：Endonuclease bamhi is evenly distributed on genome；Endonuclease bamhi has enough coverages for the coding region of gene；
Restriction enzyme site should avoid the repetitive sequence area for being distributed in genome as far as possible；Simplify efficiency suitable；Endonuclease bamhi size is suitable, fits
Together in entering performing PCR amplification and follow-up upper machine sequencing etc..
The content of the invention
The mesh of the present invention is to provide a kind of method for obtaining double DNA restricted enzyme combinations based on bioinformatics.
For achieving the above object, the technical solution used in the present invention is：
A kind of method that double DNA restricted enzyme combinations are obtained based on bioinformatics,
1) restriction enzyme site according to restricted enzyme, the double enzyme assembled schemes of design are carried out to target species reference gene group
Simulation enzyme action；
2) segment ranges produced to simulating enzyme action carry out distributional analysiss for the fragment of 90bp-750bp；
3) by its distribution situation on target species genome of above-mentioned analytic statisticss, obtain dividing for different double enzyme combinations
Cloth numerical value, the double enzyme combinations of high numerical value are optimum double enzyme combinations.
Further,
1) various pairs of DNA restricted enzyme combinations are selected, positioning chooses the enzyme action identification of combination in Concha Ostreae genome
Site, and its corresponding position coordinates on chromosome is given, according to the restriction enzyme site of restricted enzyme, the double enzyme combinations of design
Scheme is simulated enzyme action to Concha Ostreae reference gene group；
2) segment ranges produced to simulating enzyme action carry out distributional analysiss for the fragment of 90bp-750bp；
3) data obtained using above-mentioned distributional analysiss are calculated, and obtain the distribution values of different double enzyme combinations, high number
The double enzyme combinations of value are optimum double enzyme combinations.
Described pair of enzyme is combined as recognizing the combination of the restriction enzyme with the restriction enzyme for recognizing 4-5 base of 6-8 base.
The step 2) segment ranges that simulation enzyme action is produced are passed through for the fragment of 90bp-750bp
The bioinformatics software of RestrictToolKit carries out the following indexs of distributional analysiss；
Index is the simplified rate that double digestion segment ranges are 90bp-750bp, the distribution of endonuclease bamhi size, double digestion piece
Segment limit accounts for the fragment that all fragment ratios, double digestion segment ranges are 90bp-750bp for the fragment of 90bp-750bp and shows outside
The ratio of subregion, the fragment that double digestion segment ranges are 90bp-750bp ratio and double digestion segment ranges in intergenic region
For 90bp-750bp fragment unique regions distribution ratio.
The step 3) in the rule of data that obtains of distributional analysiss be：
A) for double digestion segment ranges are the simplified rate of 90bp-750bp, weight shared by which is 20%, and computing formula is：To simplify rate, y is corresponding score value to wherein x；
B) for the distribution of endonuclease bamhi size, weight shared by which is 30%, and computational methods are obtained to evaluate a rate value
Size, this rate value are equal to produced endonuclease bamhi its magnitude range and account for the ratio of whole fragments for the fragment of 90bp-750bp
Rate accounts for the ratio of whole fragments than the fragment that upper endonuclease bamhi magnitude range is below 90bp；Computing formula is：Wherein x is rate value, and y is corresponding score value；
C) for the fragment that double digestion segment ranges are 90bp-750bp accounts for the ratio of all fragments, its weight accounts for 20%,
Its computing formula is：Y=20x-4；Wherein x is the ratio that the fragment that double digestion segment ranges are 90bp-750bp accounts for all fragments
Rate, y are corresponding score value；
D) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of exon region, its weight is accounted for
10%, its computing formula is y=20x；Wherein x is that double digestion segment ranges are located at exon region for the fragment of 90bp-750bp
Ratio, y is corresponding score value；
E) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of intergenic region, its weight is accounted for
10%, its computing formula is y=-20x+14；Wherein x is that double digestion segment ranges are located between gene for the fragment of 90bp-750bp
The ratio in area, y are corresponding score value；
F) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio in unique regions, its weight is accounted for
10%, for this purpose is calculated, compared to the side on Concha Ostreae genome using BLAST softwares using the endonuclease bamhi that will be produced
Method, counts to the comparison situation of endonuclease bamhi, and computing formula is：Wherein x is double digestion fragment model
Enclose for 90bp-750bp fragment be located at unique regions ratio, y is corresponding score value；
G) calculate total score to obtain formula and be：
Wherein a is double digestion fragment model
The score value of the simplified rate for 90bp-750bp is enclosed, score value of the b for the distribution of endonuclease bamhi size, c are double digestion fragment model
The score value that the fragment for 90bp-750bp accounts for the ratio of all fragments is enclosed, d is that double digestion segment ranges are 90bp-750bp's
Fragment is located at the score value of the ratio of exon region, and e is that double digestion segment ranges are located at gene for the fragment of 90bp-750bp
Between area ratio score value, f is double digestion segment ranges is located at the ratio in unique regions for the fragment of 90bp-750bp
Score value.
Enzyme action is carried out to Concha Ostreae genomic DNA using double enzyme combinations of described optimum, with digestion products jointing, is entered
Performing PCR is expanded, fragment of the main amplification Insert Fragment for 90bp-750bp；Amplified fragments are carried out using second filial generation sequencing technologies
Sequencing.
Advantage for present invention is：
1) the enzyme action effect of arbitrarily various restriction endonuclease combinations can be predicted, and then can be according to experiment purpose from different
Restriction endonuclease is combined；
2) situation of endonuclease bamhi can be evaluated from multi-angle, many evaluation indexes, system are provided for endonuclease bamhi
Ground understands the attribute of endonuclease bamhi.
3) present invention predicts the restriction enzyme site in Concha Ostreae genome using the means of bioinformatics, by endonuclease bamhi
Parameter evaluation is carried out, optimum double restricted enzyme combinations are obtained, by double enzyme combinations of the optimum for obtaining, to Concha Ostreae genome
DNA carries out double digestion, obtains sequence information by second filial generation DNA sequencing technology, so as to simplify Concha Ostreae genome.
4) during can fast and effeciently filtering out simplified Concha Ostreae genome using the method for the present invention, suitable DNA is limited
Property enzymes double zyme combination processed, it is to avoid the blindness of work, improves work efficiency.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
1) restriction enzyme site according to restricted enzyme, selects enzyme action recognition site for the restriction enzyme of 6-8 base
Restricted enzyme of the enzyme with enzyme action recognition site for 4-5 base is combined as double enzymes；That is, DNA restriction enzymes double zymes
Combination：EcoRI and MseI, BamHI and MseI, EcoRI and HinfI, MseI and AluI；
2) the simulation endonuclease bamhi to producing is analyzed, and acquires the simulation that scope is 90bp-750bp to above-mentioned respectively
Endonuclease bamhi is analyzed：
Enzyme action is simulated to Concha Ostreae reference gene group by bioinformatics software using the combination of above-mentioned enzyme, simulation is produced
Endonuclease bamhi (sequence of the endonuclease bamhi produced after simulation enzyme action, and its coordinate position in reference gene group) is referring to table 1.
Table 1
Then the parameter of endonuclease bamhi again to producing is analyzed assessment mainly includes following parameter index：1) double digestion
Simplified rate of the segment ranges for 90-750bp；2) endonuclease bamhi size distribution；3) double digestion segment ranges are 90bp-750bp's
Fragment accounts for the ratio of all fragments；4) double digestion segment ranges are the ratio of the fragment positioned at exon region of 90bp-750bp；
5) double digestion segment ranges are the ratio of the fragment positioned at intergenic region of 90bp-750bp；6) double digestion segment ranges are 90bp-
The fragment of 750bp is located at the ratio in unique regions.
Above-mentioned parameter index calculates acquisition (referring to table 2) using by the bioinformatics software of RestrictToolKit.
The software can realize the function of simulating enzyme action, it is also possible to realize the function that the parameter of the endonuclease bamhi to producing is analyzed.
The cardinal principle of simulation enzyme action function is the lookup enzyme action recognition site in genome sequence.The parameter of the endonuclease bamhi to producing
The main Computing Principle of the function being analyzed is to calculate the fragment length summation that produces after enzyme action, and by itself and gene group leader
Degree is compared.
Table 2
A) for double digestion segment ranges are the simplified rate of 90bp-750bp, weight shared by which is 20%, and computing formula is：To simplify rate, y is corresponding score value to wherein x；
B) for the distribution of endonuclease bamhi size, weight shared by which is 30%, and computational methods are obtained to evaluate a rate value
Size, this rate value are equal to produced endonuclease bamhi its magnitude range and account for the ratio of whole fragments for the fragment of 90bp-750bp
Rate accounts for the ratio of whole fragments than the fragment that upper endonuclease bamhi magnitude range is below 90bp；Computing formula is：Wherein x is rate value, and y is corresponding score value；
C) for the fragment that double digestion segment ranges are 90bp-750bp accounts for the ratio of all fragments, its weight accounts for 20%,
Its computing formula is：Y=20x-4；Wherein x is the ratio that the fragment that double digestion segment ranges are 90bp-750bp accounts for all fragments
Rate, y are corresponding score value；
D) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of exon region, its weight is accounted for
10%, its computing formula is y=20x；Wherein x is that double digestion segment ranges are located at exon region for the fragment of 90bp-750bp
Ratio, y is corresponding score value；
E) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of intergenic region, its weight is accounted for
10%, its computing formula is y=-20x+14；Wherein x is that double digestion segment ranges are located between gene for the fragment of 90bp-750bp
The ratio in area, y are corresponding score value；
F) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio in unique regions, its weight is accounted for
10%, for this purpose is calculated, compared to the side on Concha Ostreae genome using BLAST softwares using the endonuclease bamhi that will be produced
Method, counts to the comparison situation of endonuclease bamhi, and computing formula is：Wherein x is double digestion fragment
Scope is the ratio that the fragment of 90bp-750bp is located at unique regions, and y is corresponding score value.
G) calculate total score to obtain formula and be：Its
Middle a is the score value of the simplified rate that double digestion segment ranges are 90bp-750bp, and b is the score of the distribution of endonuclease bamhi size
Value, c are the score value of the ratio that the fragment that double digestion segment ranges are 90bp-750bp accounts for all fragments, and d is double digestion fragment
Scope is the score value of the ratio that the fragment of 90bp-750bp is located at exon region, and it is 90bp- that e is double digestion segment ranges
The fragment of 750bp is located at the score value of the ratio of intergenic region, and f is that double digestion segment ranges are located at for the fragment of 90bp-750bp
The score value of the ratio in unique regions.
Table 3
4) the double enzymes for obtaining total component selections highest scoring by above-mentioned table 3 are combined, as optimum combination, using optimum combination
Enzyme action experiment is carried out to Concha Ostreae genomic DNA；That is, in table 3, optimum is combined as EcoRI and HinfI.
5) enzyme action experiment is carried out to Concha Ostreae genomic DNA using the double enzyme combinations of highest of above-mentioned acquisition；To a Concha Ostreae
Individual DNA carries out enzyme action, after the joint of the sticky end for adding the enzyme with this pair of enzyme combination, enters performing PCR amplification (main to expand
Increase fragment of the Insert Fragment for 90bp-750bp), amplification condition is 95 DEG C of 5min；{95℃10s；65℃30s；72℃30s}
15cycles；72℃5min；4℃.The DNA library of above-mentioned structure is passed through into 100PE with Illumina HiSeq2000 sequenators
Mode carry out the sequencing (sequencing depth 30x) compared with high depth.
6) above-mentioned amplified fragments are sequenced using second filial generation sequencing technologies.
The sequencing result obtained using BWA and above-mentioned software analysis sequencing data, the data that above-mentioned sequenator is produced
It is compared with prediction enzyme action result, by comparison prediction result and the effect of the difference evaluation and foreca enzyme action of sequencing result.Difference
Different situation includes：Actual sequencing result for the coverage rate of full-length genome, for gene regions, intergenic region, include sub-district, outer aobvious
The coverage rate of sub-district and enzyme action predict the outcome for full-length genome coverage rate, for gene regions, intergenic region, include sub-district,
The difference condition (referring to table 4 and table 5) of the coverage rate of exon 1.
Table 4 predicts enzyme action result
5 actual sequencing result of table
By upper table as can be seen that the prediction enzyme action result in items is consistent with actual sequencing result, in illustrating the present invention
Forecasting Methodology be very useful.
Claims (5)
1. it is a kind of that the method that double DNA restricted enzyme are combined is obtained based on bioinformatics, it is characterised in that following steps：
Step 1) according to the restriction enzyme site of restricted enzyme, the double enzyme assembled schemes of design are carried out to target species reference gene group
Simulation enzyme action；
Step 2) distributional analysiss are carried out for the fragment of 90bp-750bp to the segment ranges that simulation enzyme action is produced；
Step 3) by its distribution situation on target species genome of above-mentioned analytic statisticss, obtain dividing for different double enzyme combinations
Cloth numerical value, the double enzyme combinations of high numerical value are optimum double enzyme combinations；
The step 2) RestrictToolKit's is passed through for the fragment of 90bp-750bp to the segment ranges that simulation enzyme action is produced
Bioinformatics software carries out the following indexs of distributional analysiss；
Index is the simplified rate that double digestion segment ranges are 90bp-750bp, the distribution of endonuclease bamhi size, double digestion fragment model
Enclose the fragment for 90bp-750bp fragment that all fragment ratios, double digestion segment ranges are 90bp-750bp is accounted in exon 1
The ratio in domain, double digestion segment ranges for the fragment of 90bp-750bp in the ratio and double digestion segment ranges of intergenic region are
Distribution ratio of the fragment of 90bp-750bp in unique regions.
2. the method for obtaining double DNA restricted enzyme combinations based on bioinformatics as described in claim 1, its feature exist
In following steps：
Step 1) various pairs of DNA restricted enzyme combinations are selected, in Concha Ostreae genome, positioning chooses the enzyme action identification of combination
Site, and its corresponding position coordinates on chromosome is given, according to the restriction enzyme site of restricted enzyme, the double enzyme combinations of design
Scheme is simulated enzyme action to Concha Ostreae reference gene group；
Step 2) distributional analysiss are carried out for the fragment of 90bp-750bp to the segment ranges that simulation enzyme action is produced；
Step 3) data that obtained using above-mentioned distributional analysiss are calculated, and obtain the distribution values of different double enzymes combinations, high number
The double enzyme combinations of value are optimum double enzyme combinations.
3. the method for obtaining double DNA restricted enzyme combinations based on bioinformatics as described in claim 1 or 2, which is special
Levy and be：Described pair of enzyme is combined as recognizing the combination of the restriction enzyme with the restriction enzyme for recognizing 4-5 base of 6-8 base.
4. the method for obtaining double DNA restricted enzyme combinations based on bioinformatics as described in claim 2, its feature exist
In：
The step 3) in the rule of data that obtains of distributional analysiss be：
A) for double digestion segment ranges are the simplified rate of 90bp-750bp, weight shared by which is 20%, and computing formula is：To simplify rate, y is corresponding score value to wherein x；
B) for the distribution of endonuclease bamhi size, weight shared by which is 30%, and computational methods obtain size to evaluate a rate value,
This rate value is equal to produced endonuclease bamhi its magnitude range and accounts for the ratio ratio of whole fragments for the fragment of 90bp-750bp
Upper endonuclease bamhi magnitude range is the ratio that the fragment of below 90bp accounts for whole fragments；Computing formula is：Its
Middle x is rate value, and y is corresponding score value；
C) for the fragment that double digestion segment ranges are 90bp-750bp accounts for the ratio of all fragments, its weight accounts for 20%, its meter
Calculating formula is：Y=20x-4；Wherein x accounts for the ratio of all fragments for the fragment that double digestion segment ranges are 90bp-750bp, and y is
Corresponding score value；
D) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of exon region, its weight accounts for 10%,
Its computing formula is y=20x；Wherein x is the ratio that the fragment that double digestion segment ranges are 90bp-750bp is located at exon region
Rate, y are corresponding score value；
E) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio of intergenic region, its weight accounts for 10%, its
Computing formula is y=-20x+14；Wherein x is the ratio that the fragment that double digestion segment ranges are 90bp-750bp is located at intergenic region
Rate, y are corresponding score value；
F) for the fragment that double digestion segment ranges are 90bp-750bp is located at the ratio in unique regions, its weight accounts for 10%,
For this purpose is calculated, compared to the method on Concha Ostreae genome using BLAST softwares using the endonuclease bamhi that will be produced, it is right
The comparison situation of endonuclease bamhi is counted, and computing formula is：Wherein x for double digestion segment ranges is
The fragment of 90bp-750bp is located at the ratio in unique regions, and y is corresponding score value；
G) calculate total score to obtain formula and be：
It is 90bp- that wherein a is double digestion segment ranges
The score value of the simplified rate of 750bp, score value of the b for the distribution of endonuclease bamhi size, it is 90bp- that c is double digestion segment ranges
The fragment of 750bp accounts for the score value of the ratio of all fragments, and d is outside the fragment that double digestion segment ranges are 90bp-750bp is located at
The score value of the ratio of aobvious subregion, e are the ratio that the fragment that double digestion segment ranges are 90bp-750bp is located at intergenic region
Score value, f be double digestion segment ranges for 90bp-750bp fragment be located at unique regions ratio score value.
5. the method for obtaining double DNA restricted enzyme combinations based on bioinformatics as described in claim 1, its feature exist
In：Enzyme action is carried out to Concha Ostreae genomic DNA using double enzyme combinations of described optimum, with digestion products jointing, enters performing PCR
Amplification, expands fragment of the Insert Fragment for 90bp-750bp；Amplified fragments are sequenced using second filial generation sequencing technologies.
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| CN101251511A (en) * | 2008-03-14 | 2008-08-27 | 毅新兴业（北京）科技有限公司 | Method for testing SNP using restriction enzymes double zyme cutting |
| JP5061346B2 (en) * | 2006-12-26 | 2012-10-31 | 国立大学法人山口大学 | Method for producing DNA fragment |
| CN103740702A (en) * | 2014-01-07 | 2014-04-23 | 中国科学院海洋研究所 | SNP (Single Nucleotide Polymorphism) marker relevant to heat tolerance of argopectehs irradias and identification method and potential application thereof |
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| JP5061346B2 (en) * | 2006-12-26 | 2012-10-31 | 国立大学法人山口大学 | Method for producing DNA fragment |
| CN101251511A (en) * | 2008-03-14 | 2008-08-27 | 毅新兴业（北京）科技有限公司 | Method for testing SNP using restriction enzymes double zyme cutting |
| CN103740702A (en) * | 2014-01-07 | 2014-04-23 | 中国科学院海洋研究所 | SNP (Single Nucleotide Polymorphism) marker relevant to heat tolerance of argopectehs irradias and identification method and potential application thereof |
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| 《两种双酶切组合在细鳞鱼AFLP体系中的比较分析》;王荻 等；;《大连水产学院学报》;20091031;第24卷(第5期);第460-464页； * |
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