Computational protocol: Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF, and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

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Protocol publication

[…] Genomic DNA was isolated by a standard phenol chloroform extraction method. Seven genomic DNA samples were sequenced on Illumina HiSeq 2000 using Agilent SureSelect Human All Exon V4 51Mb enrichment kit. Sequence reads generated from the libraries were filtered for quality, aligned and mapped to the hg19 human reference genome using the gsNap program []. The variant calling process for both indels (insertion/deletions) and single nucleotide variants was done by using the Genome Analysis Toolkit (GATK, http://www.broadinstitute.org/gatk). ANNOVAR software was applied for variant functional annotation []. The deleterious impact of non-synonymous SNPs was predicted by the PolyPhen-2 (version 2.2.2) (http://genetics.bwh.harvard.edu/pph2) []. Under the assumption that HL in siblings from families F38, F40, and F54 was likely caused by the same mutations in corresponding gene with recessive pattern of inheritance, we focused our evaluation of the WES data sets only on homozygous or compound heterozygous variants shared by affected siblings from the same family. The only patient from family F53 was screened for rare deleterious variants known as related with HL according to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/). Additional criteria for filtering variants was an allele frequency < 5%. All shared variants predicted by PolyPhen-2 as “possibly damaging” or “probably damaging” were further analyzed for zygosity in the context of a candidate gene list for NSHL genes with known autosomal recessive inheritance patterns []. To extend the candidate gene list we have also searched OMIM records (http://www.omim.org/) for the terms “hearing loss” and “deafness”. Rarity of the variants was predicted based on the alternate allele frequency according to the dbSNP138 (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genomes Project (http://www.1000genomes.org/), Exome Sequencing Project (ESP, 6500 exomes, http://evs.gs.washington.edu/EVS/), and Exome Aggregation Consortium (ExAC, Cambridge, MA, http://exac.broadinstitute.org). […]

Pipeline specifications

Software tools GSNAP, GATK, ANNOVAR, PolyPhen
Databases ClinVar dbSNP OMIM
Application WES analysis
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn, Smith-Magenis Syndrome