CN104268441B - Method for obtaining double DNA restriction endonuclease combinations based on bioinformatics - Google Patents
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Abstract
Description
技术领域technical field
本发明涉及生物信息学，具体说是一种基于生物信息学获得双DNA限制性内切酶组合的方法。The invention relates to bioinformatics, in particular to a method for obtaining a combination of double DNA restriction endonucleases based on bioinformatics.
背景技术Background technique
随着第二代DNA测序技术的发展，DNA测序结果的质量不断提高，测序成本逐渐降低。很多科研工作者都在采用二代测序技术来获得待研究物种的序列信息。对于待研究个体数目较少(少于100个)的项目来讲，全基因组测序具有序列信息丰富、基因组覆盖范围广泛的优点，但是对于待研究个体数目较多(多于100个)的项目来说，全基因组重测序的成本仍然很大。如何能在保证获得足够的序列信息的基础上降低测序成本就变得非常重要了。目前主流的方法是RAD、GBS等简化基因组的方法，这两种方法简化基因组信息的主要方式就是通过DNA限制性内切酶酶切基因组DNA，通过富集带有酶切位点的片段来达到简化的目的。这种方式最重要的步骤就是选择合适的限制性内切酶，合适的限制性内切酶应该满足几个条件：酶切片段均匀分布在基因组上；酶切片段对于基因的编码区具有足够的覆盖度；酶切位点应该尽量避免分布在基因组的重复序列区；简化效率合适；酶切片段大小合适，适合于进行PCR扩增和后续的上机测序等。With the development of second-generation DNA sequencing technology, the quality of DNA sequencing results has been continuously improved, and the cost of sequencing has been gradually reduced. Many scientific researchers are using next-generation sequencing technology to obtain the sequence information of the species to be studied. For projects with a small number of individuals to be studied (less than 100), whole genome sequencing has the advantages of rich sequence information and wide genome coverage, but for projects with a large number of individuals to be studied (more than 100) That said, the cost of whole-genome resequencing remains substantial. How to reduce the cost of sequencing on the basis of ensuring sufficient sequence information becomes very important. At present, the mainstream methods are RAD, GBS and other methods to simplify the genome. The main way of these two methods to simplify the genome information is to digest genomic DNA by DNA restriction endonuclease, and enrich the fragments with restriction sites to achieve purpose of simplification. The most important step in this method is to select a suitable restriction enzyme. A suitable restriction enzyme should meet several conditions: the cleavage fragments are evenly distributed on the genome; Coverage; restriction sites should be avoided in the repetitive sequence region of the genome as much as possible; the simplification efficiency is appropriate; the restriction fragment size is appropriate, suitable for PCR amplification and subsequent sequencing on the machine.
发明内容Contents of the invention
本发明的目在于提供一种基于生物信息学获得双DNA限制性内切酶组合的方法。The object of the present invention is to provide a method for obtaining a combination of double DNA restriction endonucleases based on bioinformatics.
为实现上述目的，本发明采用的技术方案为：To achieve the above object, the technical solution adopted in the present invention is:
一种基于生物信息学获得双DNA限制性内切酶组合的方法，A method for obtaining double DNA restriction endonuclease combinations based on bioinformatics,
1)根据限制性内切酶的酶切位点，设计双酶组合方案对目标物种参考基因组进行模拟酶切；1) According to the cleavage sites of restriction endonucleases, design a dual-enzyme combination scheme to perform simulated digestion on the reference genome of the target species;
2)对模拟酶切产生的片段范围为90bp-750bp的片段进行分布分析；2) Analyze the distribution of fragments in the range of 90bp-750bp produced by simulated enzyme digestion;
3)通过上述分析统计其在目标物种基因组上的分布情况，获得不同双酶组合的分布数值，高数值双酶组合即为最优的双酶组合。3) Through the above-mentioned analysis and statistics of its distribution on the genome of the target species, the distribution values of different dual-enzyme combinations are obtained, and the high-value dual-enzyme combination is the optimal dual-enzyme combination.
进一步的说，Further,
1)选择多种双DNA限制性内切酶组合，在牡蛎基因组中定位选取组合的酶切识别位点，并给出其在染色体上相应的位置坐标，根据限制性内切酶的酶切位点，设计双酶组合方案对牡蛎参考基因组进行模拟酶切；1) Select a variety of double DNA restriction endonuclease combinations, position and select the combined restriction endonuclease recognition site in the oyster genome, and give its corresponding position coordinates on the chromosome, according to the restriction endonuclease restriction endonuclease restriction site Design a dual-enzyme combination scheme to perform simulated enzyme digestion on the oyster reference genome;
2)对模拟酶切产生的片段范围为90bp-750bp的片段进行分布分析；2) Analyze the distribution of fragments in the range of 90bp-750bp produced by simulated enzyme digestion;
3)利用上述分布分析得到的数据进行计算，获得不同双酶组合的分布数值，高数值双酶组合即为最优的双酶组合。3) Use the data obtained from the above distribution analysis to perform calculations to obtain the distribution values of different dual-enzyme combinations, and the high-value dual-enzyme combination is the optimal dual-enzyme combination.
所述双酶组合为识别6-8个碱基的限制酶与识别4-5个碱基的限制酶的组合。The double enzyme combination is a combination of restriction enzymes recognizing 6-8 bases and restriction enzymes recognizing 4-5 bases.
所述步骤2)对模拟酶切产生的片段范围为90bp-750bp的片段通过RestrictToolKit的生物信息学软件进行分布分析下述指标；The step 2) carries out distribution analysis on the fragments of the fragment range of 90bp-750bp produced by the simulated enzyme digestion through the bioinformatics software of RestrictToolKit to analyze the following indicators;
指标为双酶切片段范围为90bp-750bp的简化率、酶切片段大小的分布、双酶切片段范围为90bp-750bp的片段占所有片段比率、双酶切片段范围为90bp-750bp的片段在外显子区域的比率、双酶切片段范围为90bp-750bp的片段在基因间区的比率和双酶切片段范围为90bp-750bp的片段在unique区域的分布比率。The indicators are the simplification rate of double-digested fragments ranging from 90bp-750bp, the size distribution of enzyme-digested fragments, the ratio of double-digested fragments ranging from 90bp-750bp to all fragments, and the range of double-digested fragments ranging from 90bp-750bp is excluded The ratio of the exon region, the ratio of the double-digested fragments ranging from 90bp-750bp in the intergenic region, and the distribution ratio of the double-digested fragments ranging from 90bp-750bp in the unique region.
所述步骤3)中分布分析得到的数据的规则为：The rule of the data that distribution analysis obtains in described step 3) is:
a)对于双酶切片段范围为90bp-750bp的简化率，其所占权重为20％，计算公式为：其中x为简化率，y为相对应的得分值；a) For the simplification rate of double-digested fragments ranging from 90bp to 750bp, its weight is 20%, and the calculation formula is: Where x is the simplification rate, and y is the corresponding score value;
b)对于酶切片段大小的分布，其所占权重为30％，计算方法为评价一个比率值得大小，这个比率值等于所产生的酶切片段其大小范围为90bp-750bp的片段占全部片段的比率比上酶切片段大小范围为90bp以下的片段占全部片段的比率；计算公式为：其中x为比率值，y为相对应的得分值；b) For the distribution of restriction fragment size, its weight is 30%, and the calculation method is to evaluate a ratio value, which is equal to the fragments with a size range of 90bp-750bp in the total fragments of the generated restriction fragments Ratio Ratio: The ratio of fragments with a size range of 90 bp or less to all fragments; the calculation formula is: Where x is the ratio value and y is the corresponding score value;
c)对于双酶切片段范围为90bp-750bp的片段占所有片段的比率，其权重占20％，其计算公式为：y＝20x-4；其中x为双酶切片段范围为90bp-750bp的片段占所有片段的比率，y为相对应的得分值；c) For the ratio of fragments with a double-digestion range of 90bp-750bp to all fragments, its weight accounts for 20%, and its calculation formula is: y=20x-4; where x is the double-digestion fragment range of 90bp-750bp The ratio of fragments to all fragments, y is the corresponding score value;
d)对于双酶切片段范围为90bp-750bp的片段位于外显子区域的比率，其权重占10％，其计算公式为y＝20x；其中x为双酶切片段范围为90bp-750bp的片段位于外显子区域的比率，y为相对应的得分值；d) For the ratio of the fragments within the range of 90bp-750bp of double-digested fragments located in the exon region, its weight accounts for 10%, and the calculation formula is y=20x; where x is the fragment of double-digested fragments within the range of 90bp-750bp The ratio located in the exon region, y is the corresponding score value;
e)对于双酶切片段范围为90bp-750bp的片段位于基因间区的比率，其权重占10％，其计算公式为y＝-20x+14；其中x为双酶切片段范围为90bp-750bp的片段位于基因间区的比率，y为相对应的得分值；e) For the ratio of the double-digested fragments in the range of 90bp-750bp located in the intergenic region, its weight accounts for 10%, and its calculation formula is y=-20x+14; where x is the double-digested fragments in the range of 90bp-750bp The ratio of fragments located in the intergenic region, y is the corresponding score value;
f)对于双酶切片段范围为90bp-750bp的片段位于unique区域的比率，其权重占10％，对于此项目的计算，采用将产生的酶切片段使用BLAST软件比对到牡蛎基因组上的方法，对酶切片段的比对情况进行统计，计算公式为：其中x为双酶切片段范围为90bp-750bp的片段位于unique区域的比率，y为相对应的得分值；f) For the proportion of double-digestion fragments in the range of 90bp-750bp located in the unique region, its weight accounts for 10%. For the calculation of this project, the method of comparing the generated restriction fragments to the oyster genome using BLAST software , to carry out statistics on the comparison of enzyme-digested fragments, the calculation formula is: Where x is the ratio of the double-digested fragments in the range of 90bp-750bp located in the unique region, and y is the corresponding score value;
g)计算总分值得公式为：g) The formula for calculating the total score is:
其中a为双酶切片段范围为90bp-750bp的简化率的得分值，b为酶切片段大小的分布的得分值，c为双酶切片段范围为90bp-750bp的片段占所有片段的比率的得分值，d为双酶切片段范围为90bp-750bp的片段位于外显子区域的比率的得分值，e为双酶切片段范围为90bp-750bp的片段位于基因间区的比率的得分值，f为双酶切片段范围为90bp-750bp的片段位于unique区域的比率的得分值。 Where a is the score value of the simplification rate of the double-digested fragment range of 90bp-750bp, b is the score value of the distribution of the size of the enzyme-digested fragment, and c is the percentage of the fragments of the double-digested fragment range of 90bp-750bp in all fragments The score value of the ratio, d is the score value of the ratio of the fragments with the range of 90bp-750bp in the exon region, and e is the ratio of the fragments in the intergenic region with the range of 90bp-750bp in the double restriction fragments f is the score value of the ratio of double-digested fragments in the range of 90bp-750bp located in the unique region.
利用所述的最优的双酶组合对牡蛎基因组DNA进行酶切，以酶切产物连接接头，进行PCR扩增，主要扩增插入片段为90bp-750bp的片段；将扩增片段利用第二代测序技术进行测序。Utilize the above-mentioned optimal double-enzyme combination to digest the oyster genomic DNA, connect the adapter with the digested product, and perform PCR amplification. The main amplified insert fragment is a fragment of 90bp-750bp; Sequencing technology for sequencing.
本发明所具有的优点在于：The advantages that the present invention has are:
1)可以预测任意多种内切酶组合的酶切效果，进而可以根据实验目的选用不同的内切酶组合；1) It can predict the digestion effect of any combination of endonucleases, and then choose different combinations of endonucleases according to the purpose of the experiment;
2)可以从多角度评价酶切片段的情况，为酶切片段提供多方面的评价指标，系统地了解酶切片段的属性。2) It can evaluate the digested fragments from multiple angles, provide multiple evaluation indicators for the digested fragments, and systematically understand the properties of the digested fragments.
3)本发明采用生物信息学的手段预测牡蛎基因组中的酶切位点，通过对酶切片段进行参数评价，获得最优的双限制性内切酶组合，将获得的最优的双酶组合，对牡蛎基因组DNA进行双酶切，通过第二代DNA测序技术获得序列信息，从而简化牡蛎基因组。3) The present invention uses bioinformatics means to predict the enzyme cleavage site in the oyster genome, and obtains the optimal double restriction endonuclease combination by evaluating the parameters of the enzyme cleavage fragment, and the obtained optimal double enzyme combination , double-enzyme digestion of oyster genomic DNA, and sequence information obtained by second-generation DNA sequencing technology, thereby simplifying the oyster genome.
4)采用本发明的方法可以快速有效地筛选出简化牡蛎基因组过程中合适的DNA限制性内切酶双酶组合，避免工作的盲目性，提高工作效率。4) The method of the present invention can quickly and effectively screen out the appropriate DNA restriction endonuclease double-enzyme combination in the process of simplifying the oyster genome, avoiding blindness in work and improving work efficiency.
具体实施方式detailed description
以下实施例用于说明本发明，但不用来限制本发明的范围。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
实施例1Example 1
1)根据限制性内切酶的酶切位点，选择酶切识别位点为6-8个碱基的限制性内切酶与酶切识别位点为4-5个碱基的限制性内切酶作为双酶组合；即，DNA限制性内切酶双酶组合：EcoRI与MseI、BamHI与MseI、EcoRI与HinfI、MseI与AluI；1) According to the restriction endonuclease restriction site, select the restriction endonuclease with the recognition site of 6-8 bases and the restriction endonuclease with the recognition site of 4-5 bases. Dicer as a double-enzyme combination; that is, a DNA restriction enzyme double-enzyme combination: EcoRI and MseI, BamHI and MseI, EcoRI and HinfI, MseI and AluI;
2)对产生的模拟酶切片段进行分析，分别对上述获得到范围为90bp-750bp的模拟酶切片段进行分析：2) Analyzing the generated simulated enzyme-cut fragments, respectively analyzing the above-mentioned simulated enzyme-cut fragments in the range of 90bp-750bp:
利用上述酶组合通过生物信息学软件对牡蛎参考基因组进行模拟酶切，产生模拟酶切片段(模拟酶切后产生的酶切片段的序列，及其在参考基因组上的坐标位置)参见表1。Using the combination of enzymes above, the oyster reference genome was simulated by bioinformatics software to generate simulated digestion fragments (sequences of the digested fragments produced after simulated digestion and their coordinate positions on the reference genome) are shown in Table 1.
表1Table 1
而后再对产生的酶切片段的参数进行分析评估主要包括如下参数指标：1)双酶切片段范围为90-750bp的简化率；2)酶切片段大小分布；3)双酶切片段范围为90bp-750bp的片段占所有片段的比率；4)双酶切片段范围为90bp-750bp的片段位于外显子区域的比率；5)双酶切片段范围为90bp-750bp的片段位于基因间区的比率；6)双酶切片段范围为90bp-750bp的片段位于unique区域的比率。Then analyze and evaluate the parameters of the generated enzyme-digested fragments, mainly including the following parameters: 1) the simplification rate of double-enzyme-digested fragments ranging from 90-750bp; 2) the size distribution of enzyme-digested fragments; 3) the range of double-enzyme-digested fragments is The ratio of 90bp-750bp fragments to all fragments; 4) The ratio of double-digested fragments in the range of 90bp-750bp located in the exon region; 5) The ratio of double-digested fragments in the range of 90bp-750bp located in the intergenic region Ratio; 6) The ratio of the double-digested fragments in the range of 90bp-750bp located in the unique region.
上述参数指标采用通过RestrictToolKit的生物信息学软件计算获得(参见表2)。该软件可以实现模拟酶切的功能，也可以实现对产生的酶切片段的参数进行分析的功能。模拟酶切功能的主要原理是在基因组序列中查找酶切识别位点。对产生的酶切片段的参数进行分析的功能的主要计算原理为计算酶切之后产生的片段长度总和，并将其与基因组长度进行比较。The above parameter indexes are obtained by calculating through the bioinformatics software of RestrictToolKit (see Table 2). The software can realize the function of simulating enzyme digestion, and can also realize the function of analyzing the parameters of the generated enzyme digestion fragments. The main principle of the simulated restriction function is to find restriction recognition sites in the genome sequence. The main calculation principle of the function of analyzing the parameters of the generated restriction fragments is to calculate the sum of the lengths of the fragments generated after restriction digestion and compare it with the length of the genome.
表2Table 2
a)对于双酶切片段范围为90bp-750bp的简化率，其所占权重为20％，计算公式为：其中x为简化率，y为相对应的得分值；a) For the simplification rate of double-digested fragments ranging from 90bp to 750bp, its weight is 20%, and the calculation formula is: Where x is the simplification rate, and y is the corresponding score value;
b)对于酶切片段大小的分布，其所占权重为30％，计算方法为评价一个比率值得大小，这个比率值等于所产生的酶切片段其大小范围为90bp-750bp的片段占全部片段的比率比上酶切片段大小范围为90bp以下的片段占全部片段的比率；计算公式为：其中x为比率值，y为相对应的得分值；b) For the distribution of restriction fragment size, its weight is 30%, and the calculation method is to evaluate a ratio value, which is equal to the fragments with a size range of 90bp-750bp in the total fragments of the generated restriction fragments Ratio Ratio: The ratio of fragments with a size range of 90 bp or less to all fragments; the calculation formula is: Where x is the ratio value and y is the corresponding score value;
c)对于双酶切片段范围为90bp-750bp的片段占所有片段的比率，其权重占20％，其计算公式为：y＝20x-4；其中x为双酶切片段范围为90bp-750bp的片段占所有片段的比率，y为相对应的得分值；c) For the ratio of fragments with a double-digestion range of 90bp-750bp to all fragments, its weight accounts for 20%, and its calculation formula is: y=20x-4; where x is the double-digestion fragment range of 90bp-750bp The ratio of fragments to all fragments, y is the corresponding score value;
d)对于双酶切片段范围为90bp-750bp的片段位于外显子区域的比率，其权重占10％，其计算公式为y＝20x；其中x为双酶切片段范围为90bp-750bp的片段位于外显子区域的比率，y为相对应的得分值；d) For the ratio of the fragments within the range of 90bp-750bp of double-digested fragments located in the exon region, its weight accounts for 10%, and the calculation formula is y=20x; where x is the fragment of double-digested fragments within the range of 90bp-750bp The ratio located in the exon region, y is the corresponding score value;
e)对于双酶切片段范围为90bp-750bp的片段位于基因间区的比率，其权重占10％，其计算公式为y＝-20x+14；其中x为双酶切片段范围为90bp-750bp的片段位于基因间区的比率，y为相对应的得分值；e) For the ratio of the double-digested fragments in the range of 90bp-750bp located in the intergenic region, its weight accounts for 10%, and its calculation formula is y=-20x+14; where x is the double-digested fragments in the range of 90bp-750bp The ratio of fragments located in the intergenic region, y is the corresponding score value;
f)对于双酶切片段范围为90bp-750bp的片段位于unique区域的比率，其权重占10％，对于此项目的计算，采用将产生的酶切片段使用BLAST软件比对到牡蛎基因组上的方法，对酶切片段的比对情况进行统计，计算公式为：其中x为双酶切片段范围为90bp-750bp的片段位于unique区域的比率，y为相对应的得分值。f) For the proportion of double-digestion fragments in the range of 90bp-750bp located in the unique region, its weight accounts for 10%. For the calculation of this project, the method of comparing the generated restriction fragments to the oyster genome using BLAST software , to carry out statistics on the comparison of enzyme-digested fragments, the calculation formula is: Where x is the ratio of double-digested fragments in the range of 90bp-750bp located in the unique region, and y is the corresponding score value.
g)计算总分值得公式为：其中a为双酶切片段范围为90bp-750bp的简化率的得分值，b为酶切片段大小的分布的得分值，c为双酶切片段范围为90bp-750bp的片段占所有片段的比率的得分值，d为双酶切片段范围为90bp-750bp的片段位于外显子区域的比率的得分值，e为双酶切片段范围为90bp-750bp的片段位于基因间区的比率的得分值，f为双酶切片段范围为90bp-750bp的片段位于unique区域的比率的得分值。g) The formula for calculating the total score is: Where a is the score value of the simplification rate of the double-digested fragment range of 90bp-750bp, b is the score value of the distribution of the size of the enzyme-digested fragment, and c is the percentage of the fragments of the double-digested fragment range of 90bp-750bp in all fragments The score value of the ratio, d is the score value of the ratio of the fragments with the range of 90bp-750bp in the exon region, and e is the ratio of the fragments in the intergenic region with the range of 90bp-750bp in the double restriction fragments f is the score value of the ratio of double-digested fragments in the range of 90bp-750bp located in the unique region.
表3table 3
4)由上述表3获得总分选择得分最高的双酶组合，即为最优的组合，利用最优组合对牡蛎基因组DNA进行酶切实验；即，表3中最优的组合为EcoRI与HinfI。4) Obtain the total score obtained from the above table 3 and select the dual enzyme combination with the highest score, which is the optimal combination, and use the optimal combination to carry out enzyme digestion experiments on oyster genomic DNA; that is, the optimal combination in Table 3 is EcoRI and HinfI .
5)利用上述获得的最高的双酶组合对牡蛎基因组DNA进行酶切实验；对一个牡蛎个体DNA进行酶切，加上带有这种双酶组合的酶的粘性末端的接头后，进行PCR扩增(主要扩增插入片段为90bp-750bp的片段)，扩增条件为95℃5min；{95℃10s；65℃30s；72℃30s}15cycles；72℃5min；4℃。将上述构建的DNA文库用Illumina HiSeq2000测序仪通过100PE的方式进行较高深度的测序(测序深度30x)。5) Utilize the highest double-enzyme combination obtained above to perform an enzyme digestion experiment on oyster genomic DNA; carry out enzyme digestion to an oyster individual DNA, add the adapter with the sticky end of the enzyme of this double-enzyme combination, and then perform PCR amplification. Amplification (the main amplified insert fragment is a fragment of 90bp-750bp), the amplification conditions are 95°C 5min; {95°C 10s; 65°C 30s; 72°C 30s} 15cycles; 72°C 5min; 4°C. The DNA library constructed above was sequenced at a higher depth (sequencing depth 30x) by means of 100PE using an Illumina HiSeq2000 sequencer.
6)将上述扩增片段利用第二代测序技术进行测序。6) Sequencing the amplified fragments above using second-generation sequencing technology.
利用BWA以及上述软件分析测序数据，将上述测序仪产生的数据获得的测序结果与预测酶切结果进行比较，通过比较预测结果与测序结果的差异评价预测酶切的效果。差异情况包括：实际测序结果对于全基因组的覆盖率、对于基因区、基因间区、内含子区、外显子区的覆盖率与酶切预测结果对于全基因组的覆盖率、对于基因区、基因间区、内含子区、外显子区的覆盖率的差异情况(参见表4和表5)。Use BWA and the above-mentioned software to analyze the sequencing data, compare the sequencing results obtained from the data generated by the above-mentioned sequencer with the predicted enzyme digestion results, and evaluate the effect of the predicted enzyme digestion by comparing the difference between the predicted results and the sequencing results. Differences include: the coverage of the actual sequencing results for the whole genome, the coverage of gene regions, intergenic regions, intron regions, and exon regions, and the coverage of the enzyme digestion prediction results for the whole genome, for gene regions, Differences in coverage of intergenic regions, intron regions, and exon regions (see Table 4 and Table 5).
表4预测酶切结果Table 4 predicted enzyme digestion results
表5实际测序结果Table 5 Actual sequencing results
通过上表可以看出，各项中的预测酶切结果与实际测序结果相符，说明本发明中的预测方法是非常实用的。It can be seen from the above table that the predicted enzyme digestion results in each item are consistent with the actual sequencing results, indicating that the prediction method in the present invention is very practical.
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