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

作者：Oliveira Theo G M^*; Mitne Neto Miguel; Cerdeira Louise T; Marsiglia Julia D C; Arteaga Fernandez Edmundo; Krieger Jose E; Pereira Alexandre C

来源：Journal of Molecular Diagnostics, 2015, 17(4): 420-430.

DOI：10.1016/j.jmoldx.2015.02.003

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 250x (95% CI, 226.03-273.91) and 95.2% of target bases with a coverage of >= 10x. 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 reproducibility values were 89.5% and 87.3%, respectively. After a careful assessment of analytical performance, we infer that the assay presents potential feasibility for application in diagnostic routines, with minimal time requirements and a simple bioinformatics structure.

• 出版日期2015-7