Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.
Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift.
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DOI：
10.3389/fgene.2012.00035
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发表时间：
2012
影响因子：
3.7
通讯作者：
Lu X
中科院分区：
文献类型：
--
作者：
Cingolani P;Patel VM;Coon M;Nguyen T;Land SJ;Ruden DM;Lu X
本文介绍了一种新的程序SnpSift过滤差异DNA序列变异后，遗传毒性化学品暴露在两个或两个以上的实验基因组。在这里，我们说明了如何SnpSift可以用来识别候选表型相关的变体，包括单核苷酸多态性，多核苷酸多态性，插入和缺失（InDel）的突变株分离的全基因组化学诱变的果蝇。首先，使用Illumina下一代DNA测序仪对两种独立分离的突变蝇株的基因组进行测序，以获得20- 29倍的常染色质序列覆盖率，所述突变蝇株是由遗传毒性化学品暴露产生的新型隐性雄性不育基因座的等位基因。处理测序读数，并使用标准生物信息学工具调用变体。接下来，使用SnpEff注释所有序列变体及其对相关基因的潜在突变效应。然后，使用SnpSift来过滤和选择可能破坏两个等位基因突变株中的共同基因的差异变体。通过聚合酶链反应扩增DNA的毛细管测序，部分验证了潜在的致病性DNA病变的遗传间隔定义的减数分裂映射和删除染色体的定义区域的删除。在位于遗传区间的五个候选基因中，发现PKa样基因CG 12069在两个等位基因突变体中的每一个中携带单独的早熟终止密码子突变，而在该区间内的其他四个候选基因具有野生型序列。因此，Pka类基因是雄性不育基因座的强候选基因。这些结果表明，组合SnpEff和SnpSift可以加快鉴定化学诱变的果蝇品系中的候选表型致病突变。该技术也可用于表征遗传毒性化学品产生的各种突变。
This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions, and deletions (InDels) in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of polymerase chain reaction-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate pre-mature stop codon mutation in each of the two allelic mutants whereas the other four candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic chemicals.