Identification and validation of loss of function variants in clinical contexts

The choice of an appropriate variant calling pipeline for exome sequencing data is becoming increasingly more important in translational medicine projects and clinical contexts. Within GOSgene, which facilitates genetic analysis as part of a joint effort of the University College London and the Great Ormond Street Hospital, we aimed to optimize a variant calling pipeline suitable for our clini- cal context. We implemented the GATK/Queue framework and evaluated the performance of its two callers: the classical UnifiedGenotyper and the new vari- ant discovery tool HaplotypeCaller. We performed an experimental validation of the loss-of-function (LoF) variants called by the two methods using Seque- nom technology. UnifiedGenotyper showed a total validation rate of 97.6% for LoF single-nucleotide polymorphisms (SNPs) and 92.0% for insertions or dele- tions (INDELs), whereas HaplotypeCaller was 91.7% for SNPs and 55.9% for INDELs. We confirm that GATK/Queue is a reliable pipeline in translational medicine and clinical context. We conclude that in our working environment, UnifiedGenotyper is the caller of choice, being an accurate method, with a high validation rate of error-prone calls like LoF variants. We finally highlight the importance of experimental validation, especially for INDELs, as part of a stan- dard pipeline in clinical environments.