Computational protocol: Whole exome sequencing and the clinician: we need clinical skills and functional validation in variant filtering

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Protocol publication

[…] We assessed the patient’s clinical history, examination and serological, neurophysiological, radiological and histological investigations. Exome sequencing was carried out using genomic blood DNA by Illumina Truseq™ 62 Mb exome capture. We used our in-house bioinformatics pipeline to align data to the reference human genome (UCSC hg19), remove duplicate sequence reads (Picard v1.85) and variant detection (Varscan v2.29, Dindel v1.0110). Results were filtered for variants with a minor allele frequency less than 0.01 in several databases: dbSNP135, 1000 genomes (February 2012 data release), the National Heart, Lung and Blood Institute (NHLBI, NIH, Bethesda, MD) Exome Sequencing Project (ESP) 6500 exomes, and 238 unrelated in-house controls. Rare homozygous and compound heterozygous variants were defined, and protein altering and/or putative ‘disease-causing’ mutations, along with their functional annotation, were identified using ANNOVAR. Further filtering has been performed by gene ontology (GO) terms associated with neuronal function, and Online Mendelian Inheritance in Man (OMIM) disease descriptions related to neuropathy, ataxia or extrapyramidal disorders (i.e., the patient’s clinical phenotype). We carried out PCR (IMMOLASE™ DNA Polymerase, Bioline UK) and Sanger sequencing (BigDye® Terminator v3.1) of variants predicted to be deleterious by five online prediction tools (MutationTaster, SIFT, Polyphen2, A-GVGD, and LRT). […]

Pipeline specifications

Software tools Picard, VarScan, Dindel, ANNOVAR, MutationTaster, PolyPhen
Databases dbSNP OMIM
Application WES analysis
Organisms Homo sapiens
Diseases Amyotrophic Lateral Sclerosis, Basal Ganglia Diseases, Nervous System Diseases, Parkinson Disease, Secondary, Peripheral Nervous System Diseases, Spastic Paraplegia, Hereditary