Computational protocol: Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9 mediated homology directed repair

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Protocol publication

[…] Genomic DNA was prepared from the spleen of the F0 mouse that gave rise to the ‘repaired’ line that underwent auditory phenotyping. Phenol-chloroform extracted DNA was assessed using a NanoDrop (Thermo Scientific), Epoch Microplate Spectrophotometer (Bio-Tek) and by electrophoresis on 1.5 % agarose gel containing ethidium bromide (Fisher Scientific). Whole genome sequencing (WGS) was performed using an Illumina HiSeq 2000 Sequencer. The sequence was mapped using: the BWA-mem aligner with default parameters, v.0.7.10; mouse genome reference mm10 from UCSC (original GRCm38 from NCBI, January 2012); base Phred quality cutoff, NA; keep duplicate reads, no; variable read length support, yes; and, realign gaps, no. Sequence variants were called by The Genome Analysis Toolkit (GATK) []. The BAM file was realigned for indel calling by Indel realigner. Indels were called by GATK’s HaplotypeCaller and single nucleotide variants (SNVs) were called by GATK’s UnifiedGenotyper, both using dbSNP version 137 as the background single nucleotide polymorphism (SNP) set. SNV annotations were done using NGS-SNP []. Indels were annotated using The Variant Effect Predictor [].SNVs and indels were then compared against the precompiled list found in 18 inbred strains from the Mouse Genome Project []. Further filtering was done by comparing the novel sequence variations with a wild-type (WT) C57BL/6NTac mouse genome and other in-house mouse sequences. Only high confidence sequence variations were considered in this study. High confidence SNVs and indels refer to those with Phred base quality >150 and read depth >3.Coding SNVs were investigated by PCR amplification (primers are listed in Additional file : Table S1) and Sanger sequencing of DNA from the F0 and four C57BL/6NTac WT animals, including that of a stud male employed to produce the microinjected embryos. […]

Pipeline specifications

Software tools BWA, GATK, NGS-SNP, VEP
Application WGS analysis
Organisms Mus musculus
Diseases Graft vs Host Disease