Computational protocol: Novel and De Novo Mutations Extend Association of POU3F4 with Distinct Clinical and Radiological Phenotype of Hearing Loss

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Protocol publication

[…] WES was performed on Illumina HiSeq 1500 platform (Illumina Inc., San Diego, CA, USA). Library was prepared with TruSeq Exome Enrichment kit (Illumina Inc.) according to the manufacturer’s protocol. The samples were run on 1/4 of a lane on HiSeq 1500 using 2x100 bp paired-end reads and sequenced so that 90% of target was covered 20x or more. The data were analyzed as described previously [], Briefly, raw HiSeq reads were transformed to FASTQ files using bcl2fastq software. After the adapter trimming and removal of low quality data, reads were aligned to the human genome using BWA software. After duplicate marking, local realignment and reads recalibration step, the variant calling was performed using the Haplotype Caller from GATK framework, followed by variant annotation using several databases, including EXaC, dbSNP, dbNSFP, 1000genomes and in-house variant frequencies database. For further analysis only variants with low population frequency (<1%) were considered. In the first stage of the analysis we looked at genes reported to date as associated with HL. In the subsequent step we performed wide, unfocused, analysis of rare, homozygous or compound heterozygous variants. The data from next-generation sequencing (WES) in the context of Polish law can be used for personal identification. Due to this we do not have permission to deposit such data in public databases, but data can be made available upon request to the corresponding authors. Population frequencies of POU3F4 variants were obtained from the database of the Exome Aggregation Consortium (ExAC) ( (accessed 04/2016) and the in-house database of WES data from the Polish population (n = 816). For pathogenicity prediction of the p.Val324Asp mutation the following algorithms were used: SIFT MutationTaster2, PolyPhen2 and Provean. […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, BWA, GATK, MutationTaster, PolyPhen
Databases dbNSFP dbSNP
Application WES analysis
Organisms Homo sapiens
Diseases Abnormalities, Drug-Induced