How to Surpace the wes tsunami of variants: The importance of the human factor

Abstract

Dystrophinopathies are neuromuscular X-linked recessive diseases caused by mutations in the DMD gene. Molecular alterations in this gene are large deletions/duplications in 80% of cases, identified by MLPA, and small mutations in the remaining 20%, detected by whole exome sequencing (WES). The use of next generation sequencing (NGS) techniques generates a large quantity of data that is analyzed by a bioinformatics pipeline. However, this analysis can lead to errors in the variant calling. The present work aims to emphasize the importance of the human factor in order to detect these errors. A cohort of 106 patients with presumptive clinical diagnosis of dystrophinopathy and negative MLPA results was analyzed by WES. Raw data was evaluated using the Integrative Genomics Viewer (IGV) software. Sanger sequencing was used to corroborate the identified variants. Two cases have been selected as an example to illustrate variant calling errors. Even though the WES technique and its bioinformatic pipeline proved to be fruitful, allowing us to identified pathogenic variants in muscular dystrophy genes in 91,5% of patients, we detected 2 variant calling errors among the studied individuals. In other words, the VCF results did not resemble the alteration observed in the raw data analysis. These discordances were due to the presence of deletions in the DMD gene, which caused problems in the alignment process. In both cases, alignment and annotation had to be manually re performed. While one of the patients carries a small delins, the other one has a complex rearrangement, a deletion and a 20pb insertion in the same allele. Specific primers were design to corroborate these findings.Finally, this work highlight the importance of analyzing the NGS raw data, corroborating the identified mutations by an alternative technique and the expertise of the scientist in charge of the study, so as to detect the occurrence of variant calling errors and provide reliable results to the patient.