PRJNA279479
Pilot study into whole genome resequencing of hemiclonal Drosophila melanogaster
As part of a planned study on sex-dependent genetic effects on evolutionary fitness, we sequenced the whole genomes of four female Drosophila melanogaster: One individual from the reference genome strain (Bloomington Drosophila Stock Center no. 2057); three individuals from 3 different hemiclonal lines expressed in a reference genome strain background (see Rice et al 2005, Abbott & Morrow 2011). The three hemiclonal genotypes were sampled from the LHM base population (Rice et al 2005).  Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), with volumes scaled-down according to manufacturer’s instructions. Tissue homogenisation was achieved via manual use of a microtube pestle, followed by 30 minute incubation proteinase K with regular shaking. Barrier pipette tips were used throughout, in order to minimise cross-contamination of DNA.  Sequencing was out-sourced to the University of Exeter Sequencing Service (UK, http://ess-wiki.exeter.ac.uk/ess/Main_Page). Template assessment using the Qubit BR assay (Thermo Fischer, NY, USA) indicated double-stranded DNA, 10,380bp in length at concentrations of 2-4 ng/µl. Shearing of DNA by sonication was optimised for species and low concentration to generate 200-500bp fragments. Libraries were prepared using the NEBNext DNA Ultra Library Prep Kit (New England Biolabs, MA, USA) with scaled-down volumes, and indexed using NEBNext adaptors.  All four samples were sequenced in a single lane of a HiSeq 2000 (Illumina). After removal of PhiX adaptor sequences each sample generated 32.9-41.8 million reads. Pre-filtering quality assessement indicated sequence lengths of 100bp, minimal contamination from non-Drosophilia species, 40% GC content (as expected for Drosophila melanogaster), and obtaining a ‘pass’ for per base sequence quality and per sequence quality scores (FastQC v0.10.0, http://www.bioinformatics.babraham.ac.uk/).  References 1. Abbott, J. K. & Morrow, E. H. Obtaining snapshots of genetic variation using hemiclonal analysis. Trends Ecol. Evol. 26, 359–368 (2011). 2. Rice, W. et al. Inter-locus antagonistic coevolution as an engine of speciation: Assessment with hemiclonal analysis. Proc. Natl. Acad. Sci. U. S. A. 102, 6527–6534 (2005).