A Variant Detection Pipeline for Inherited Cardiomyopathy–Associated Genes Using Next-Generation Sequencing

Publication date: July 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 4 Author(s): Théo G.M. Oliveira , Miguel Mitne-Neto , Louise T. Cerdeira , Julia D.C. Marsiglia , Edmundo Arteaga-Fernandez , José E. Krieger , Alexandre C. Pereira In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline constructed with a target panel of 74 cardiomyopathy-related genes sequenced using a next-generation sequencing system. Fifty-two samples from a hypertrophic cardiomyopathy casuistic with previous molecular diagnostics (Sanger-sequenced for MYH7, MYBCP3, and TNNT2; 19 positives and 33 negatives) were processed in parallel with a HapMap reference sample (NA12878) applied for a complete panel assessment. Sequencing coverage values were satisfactory, with a mean of 250× (95% CI, 226.03–273.91) and 95.2% of target bases with a coverage of ≥10×. With a total of 567 variants, variant call sensitivity was tested in five scenarios of coverage and variant allele frequency cutoffs. Maximum achieved sensitivity was 96.7% for single-nucleotide variants and 28.5% for indels, and positive predictive values remained above 0.959 during the whole process. Inter- and intra-assay...
Source: The Journal of Molecular Diagnostics - Category: Pathology Source Type: research