Abstract
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.
Highlights
There are two types of chemicals that cause developmental abnormities in organisms – genotoxic chemicals and non-genotoxic chemicals
In this paper, we show that SnpEff and SnpSift can be used to identify causative single nucleotide polymorphisms (SNPs) in ethyl methane sulfonate (EMS)-generated alleles of a new male-sterile mutant locus that we isolated from random chemical mutagenesis screens
The SNPs were confirmed by polymerase chain reaction (PCR) amplification and capillary sequencing and further genetic mapping of the mutant locus using overlapping chromosomal deletions
Summary
There are two types of chemicals that cause developmental abnormities in organisms – genotoxic chemicals and non-genotoxic chemicals. Most non-genotoxic chemicals only affect development or the health of the organism exposed, but some non-genotoxic chemicals such as the estrogenic chemical diethylstilbestrol (DES) can cause developmental abnormalities and increased susceptibility to cancer for several generations (reviewed in Ruden et al, 2005). Deficiencies that delete defined regions of the chromosome, typically tens to hundreds of kilobases long, can be used to further refine the boundaries of the mutated gene locus (Parks et al, 2004; Ryder et al, 2007) These positional cloning techniques are labor-intensive and time consuming, and without a guarantee of success. This frequently leads to inevitable delays in molecular and functional characterization of the gene involved, even in the post genomic era
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