Whole genome sequencing of Mus musculus: recombinant ES cells derived from the S18 line (SPRET x BL6)F1
Individual ES cell clones chosen for likely recombination that confers 6-TG resistance were picked for sequencing. Sequencing libraries for high-throughput sequencing were generated using Nextera DNA Library Prep Kit according to manufacturerŐs recommendations or using equivalent Tn5 transposase expressed in-house as previously described in Picelli et al., 2014 doi: 10.1101/gr.177881.114. Briefly, genomic DNA was extracted from FACS-sorted clones, single colonies or pooled samples by standard Protease K digestion followed by AmpureXP bead purification. Extracted high-molecular weight DNA was ŇtagmentedÓ by commercial or purified Tn5-transposase. Each tagmented DNA sample was then PCR amplified with Q5 High-Fidelity DNA Polymerase using barcoded i7-index primer (N701-N763) and the N501 i5-index primer. Pooled libraries were sequenced by a HiSeq (Illumina) at the Genome Core Facility at the MPI Tu_bingen Campus. Sequenced data were processed using a custom pipeline consisting of data clean-up, mapping, base-calling and analysis based upon fastQC v0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/); trimmomatic v0.33 (Bolger et al., 2014 doi: 10.1093/bioinformatics/btu170); bwa v0.7.10-r789 (Li and Durbin, 2010 doi: 10.1093/bioinformatics/btp698); GATK v3.4-0-gf196186 modules MarkDuplicates and IndelRealignment (McKenna et al., 2010 doi: 10.1101/gr.107524.110 and DePristo et al., 2011 doi: 10.1038/ng.806); samtools v1.2 (Li et al., 2009 doi: 10.1093/bioinformatics/btp352, Li et al., 2011 doi: 10.1093/bioinformatics/btr509.); bcftools v1.2 (https://samtools.github.io/bcftools/call-m.pdf). Genotype calls were performed against known informative single and multiple nucleotide variants between C57BL/6NJ, CAST/EiJ and SPRET/EiJ strains made available by the Wellcome Trust Sanger Centre (Mouse Genomes Project version 3 dbSNP v137 release (http://mice-geval.sanger.ac.uk/SPRET_EiJ_R20150909/Info/Index)
SUB2765932
S18_IVR_10d_6TG_resistant_30