Blog
About

57
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Related collections

          Most cited references 22

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          The variant call format and VCFtools

          Summary: The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API. Availability: http://vcftools.sourceforge.net Contact: rd@sanger.ac.uk
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.

            5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC) GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position. The periodical occurrence of G (in positions -3, -6, -9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5'-noncoding sequences is discussed.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.

              In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.
                Bookmark

                Author and article information

                Journal
                Front Genet
                Front. Gene.
                Frontiers in Genetics
                Frontiers Research Foundation
                1664-8021
                15 March 2012
                2012
                : 3
                Affiliations
                1simpleInstitute of Environmental Health Sciences, Wayne State University Detroit, MI, USA
                2simpleDepartment of Obstetrics and Gynecology, Wayne State University School of Medicine, C.S. Mott Center Detroit, MI, USA
                3simpleSchool of Computer Science and Genome Quebec Innovation Centre, McGill University Montreal, QC, Canada
                4simpleDepartment of Computer Sciences, Wayne State University Detroit, MI, USA
                Author notes

                Edited by: Michael Aschner, Vanderbilt University Medical Center, USA

                Reviewed by: Michael Aschner, Vanderbilt University Medical Center, USA; Stephen Sturzenbaum, King’s College London, UK

                *Correspondence: Douglas M. Ruden, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, C.S. Mott Center, Detroit, MI 48201, USA. e-mail: douglasr@ 123456wayne.edu

                This article was submitted to Frontiers in Toxicogenomics, a specialty of Frontiers in Genetics.

                Article
                10.3389/fgene.2012.00035
                3304048
                22435069
                Copyright © 2012 Cingolani, Patel, Coon, Nguyen, Land, Ruden and Lu.

                This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.

                Page count
                Figures: 4, Tables: 3, Equations: 0, References: 21, Pages: 9, Words: 42446
                Categories
                Genetics
                Original Research

                Comments

                Comment on this article