On the critical evaluation and confirmation of germline sequence variants identified using massively parallel sequencing

Publication date: Available online 15 April 2019Source: Journal of BiotechnologyAuthor(s): Zuzana Kubiritova, Marianna Gyuraszova, Emilia Nagyova, Michaela Hyblova, Maria Harsanyova, Jaroslav Budis, Rastislav Hekel, Juraj Gazdarica, Frantisek Duris, Ludevit Kadasi, Tomas Szemes, Jan RadvanszkyAbstractAlthough massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes. Detailed examination of false-positive and false-negative findings revealed typical error sources connected to low read depth/coverage, incomplete reference genome, indel realignment problems, as well as microsatellite associated amplification errors leading to base miss-calling. However, all these error types were identifiable with thorough manual revision of aligned reads according to specific patterns of distributions of variants and their corresponding reads. Moreover, our results point to dependence of both basic quantitative metrics (such as total read counts, alternative al...
Source: Journal of Biotechnology - Category: Biotechnology Source Type: research