Computational protocol: Mutation in WDR4 impairs tRNA m7G46 methylation and causes a distinct form of microcephalic primordial dwarfism

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Protocol publication

[…] Autozygosity mapping was performed as described before []. Briefly, genomewide genotyping using Axiom SNP chip array was followed by mapping of runs of homozygosity (ROH) >2 Mb in size as surrogates of autozygosity using AutoSNPa [], which also delineates the haplotype structure thus allowing the detection of haplotype sharing across families. Exome capture was performed using TruSeq Exome Enrichment kit (Illumina) following the manufacturer’s protocol. Samples were prepared as an Illumina sequencing library, and in the second step, the sequencing libraries were enriched for the desired target using the Illumina Exome Enrichment protocol. The captured libraries were sequenced using Illumina HiSeq 2000 Sequencer. The reads were mapped against UCSC hg19 (http://genome.ucsc.edu/) by BWA (http://bio-bwa.sourceforge.net/). The SNPs and Indels were detected by SAMTOOLS (http://samtools.sourceforge.net/). Variants from WES were filtered such that only novel (or very low frequency 0.1 %), coding/splicing, homozygous variants that are within the autozygome of the affected fetus and are predicted to be pathogenic were considered as likely causal variants []. Frequency of variants was determined using publically available variant databases (1000 Genomes, Exome Variant Server, and ExAC) as well as a database of 630 in-house ethnically-matched exomes. Pathogenicity was likely if the mutation is loss of function (splicing/truncating) or, in the case of missense/in-frame indels, removes a highly conserved amino acid and is predicted to be pathogenic by the three in silico prediction modules PolyPhen, SIFT, and CADD. […]

Pipeline specifications

Software tools BWA, SAMtools, PolyPhen
Databases Exome Variant Server
Application WES analysis
Organisms Saccharomyces cerevisiae, Homo sapiens
Diseases Congenital Abnormalities, Brain Diseases, Microcephaly