Computational protocol: A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems

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Protocol publication

[…] DNA was extracted from livers of a frogleg homozygous mutant and a wild-type littermate by standard protocols. Size selected genomic fragment libraries were prepared using the IonXpress Plus fragment library kit according to the manufacturer’s protocol, with enzymatic fragmentation performed for 12 ½ minutes at 37°C and size selection performed on a Pippen Prep set to collect fragments in the 280-342bp range. Libraries were quantitated by qPCR using the Ion Library Quantitation kit (Thermo Fisher Scientific), templated using the Ion PI template OT2 200 Kitv2 and whole genome sequencing performed on the Ion Proton platform (Thermo Fisher Scientific) using PI v2 chips and Ion PI sequencing 200 chemistry. Combining data from 3 runs for each sample the frogleg mutant and WT samples were sequenced to a depth of 10.4x and 11.8x respectively. The reads were mapped to UCSC rn5 with tmap-f3, and variants called with the TVC plugin in Torrent Suite. The genotypes at the variant positions were called by a pipeline consisting of mpileup function within samtools (version 1.2) along with calls function and vcfutils.pl in bcftools (version 1.2). AB genotypes were generated by parsing the counts in the VCF files. The variant functional annotation was performed with an in-house tool (SNP.to.ucsc-functions.pl) and Ensembl's VEP. […]

Pipeline specifications

Software tools SAMtools, bcftools
Application WGS analysis
Organisms Rattus norvegicus, Mus musculus, Homo sapiens
Diseases Infertility, Peripheral Nervous System Diseases, Limb Deformities, Congenital
Chemicals Amino Acids