A Variant Detection Pipeline for Inherited Cardiomyopathy-Associated Genes Using Next-Generation Sequencing.
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 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.
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil. Electronic address: theo.gremen@usp.br.
Théo G M
TG
Oliveira
Department of Research and Development, Fleury Group, São Paulo, Brazil.
Miguel
M
Mitne-Neto
Department of Research and Development, Fleury Group, São Paulo, Brazil.
Louise T
LT
Cerdeira
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil.
Julia D C
JD
Marsiglia
Clinical Unit of Cardiomyopathies, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil.
Edmundo
E
Arteaga-Fernandez
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil.
José E
JE
Krieger
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo, São Paulo, Brazil.
Alexandre C
AC
Pereira
D015415:Biomarkers / Q000032:analysis* / Q000378:metabolism; D024722:Cardiac Myosins / Q000235:genetics; D009202:Cardiomyopathies / Q000175:diagnosis* / Q000235:genetics; D019295:Computational Biology; D005820:Genetic Testing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D018995:Myosin Heavy Chains / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D020107:Troponin T / Q000235:genetics
D015415:Biomarkers; C501641:MYH7 protein, human; C545568:TNNT2 protein, human; D020107:Troponin T; D024722:Cardiac Myosins; D018995:Myosin Heavy Chains