Dataset Information

Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.

ABSTRACT: Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing approximately 70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.

SUBMITTER: Blumenstiel JP

PROVIDER: S-EPMC2674820 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.

Blumenstiel Justin P JP Noll Aaron C AC Griffiths Jennifer A JA Perera Anoja G AG Walton Kendra N KN Gilliland William D WD Hawley R Scott RS Staehling-Hampton Karen K

Genetics 20090323 1

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mut ...[more]

PMID: 19307605

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Project description:Pseudogenes are copies of genes that cannot produce a protein. They can be detected from disruptions to their apparent coding sequence, caused by frameshifts and premature stop codons. They are classed as either processed pseudogenes (made by reverse transcription from an mRNA) or duplicated pseudogenes, arising from duplication in the genomic DNA and subsequent disablement. Historically, there is anecdotal evidence that the fruit fly (Drosophila melanogaster) has few pseudogenes. Investigators have linked this to a high deletion rate of genomic DNA, for which there is evidence from genetic experiments on genome size. Here, we apply a homology-based pipeline that was developed previously to identify pseudogenes in other eukaryotic genomes, to the fruit fly, so as to derive the first complete survey of its pseudogene population. We find approximately 100 pseudogenes, with at least a sixth of these as candidate processed pseudogenes. This gives a much lower proportion of pseudogenes (compared with the size of the proteome) than in the genomes of other eukaryotes for which data are available (human, nematode and budding yeast). Closest matching proteins to Drosophila pseudogenes are significantly longer than the average protein in its proteome (up to approximately 60% more than the average protein's length), in contrast to the situation in the three other eukaryotic genomes. This may be due to the persistence of fragments of longer genes. In the fly pseudogene population, we found most pseudogenes for serine proteases (which are more abundant in the Drosophila lineage compared with the other eukaryotes), immunoglobulin-motif-containing proteins and cytochromes P450. Data on the sequences and positions of the putative pseudogenes are available at: http://www.pseudogene.org/fly. The detection of a small number of pseudogenes in the Drosophila genome and the higher mean length for the closest matching proteins to pseudogenes (possibly because remnants of genes encoding longer proteins are more likely to persist) are further evidence for a high deletion rate of genomic DNA in the fruit fly. The data are useful for molecular evolution study in Drosophila.

Dataset Information

Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.

Publications

Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.

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