Computational protocol: Diagnostic Yield of Whole Exome Sequencing in Pediatric Dilated Cardiomyopathy

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Protocol publication

[…] WES and variant annotation were performed on DNA samples from 21 children with idiopathic DCM (mean age at diagnosis, five years) and their unaffected parents (mean age at screening echocardiogram, 39 years), utilizing the Mayo Clinic Medical Genome Facility and Bioinformatics Core. The Agilent SureSelect Human All Exon capture kit (v2: DC-17, 45, 62, 68, 69, 79, 82; v4+UTRs: DC-80, 84, 86, 87; v5+UTRs: DC-70, 90, 92, 94, 95, 97, 101; Agilent) was utilized for exome capture. Paired-end 101 base pair sequencing was performed on Illumina’s HiSeq2000 platform (Illumina, San Diego, CA, USA). A quality control analysis of a comprehensive list of 55 known DCM genes in DC-82 revealed that, on average, over 96% of known DCM genes had 20X coverage. Reads were aligned to the hg19 reference genome with Novoalign ( followed by sorting and marking of duplicate reads using Picard ( Local realignment of insertions/deletions (INDELs) and base quality score recalibration were then performed using the Genome Analysis Toolkit (GATK) []. Single nucleotide variants (SNVs) and INDELs were called across family units simultaneously using GATK’s UnifiedGenotyper with variant quality score recalibration []. The resultant variant call format files were analyzed with Ingenuity® Variant AnalysisTM software (QIAGEN, Redwood City, CA, USA). For families DC-68 and DC-80, each of the two affected children was required to carry a variant for it to pass filtering criteria. To determine rarity of variants, minor allele frequencies from three publicly available population databases were used: 1000 Genomes (1000 G, WGS data from 1092 individuals) []; European individuals in Exome Variant Server (EVS, WES data from 4300 individuals) []; or European individuals in the Exome Aggregation Consortium (ExAC, WES data from 33,370 individuals) []. Because TTN is highly tolerant of single nucleotide variation (RVIS_ExAC = 99.5%) [] and rare missense variants are common in healthy controls, missense variants in this gene were excluded from the analyses []. Sanger sequencing was performed to confirm pathogenic variants, with primer pairs listed in . In silico tools were utilized to predict the biological impact of identified mutations. We employed Combined Annotation-Dependent Depletion (CADD) scores [] for missense mutations, and probability of loss-of-function intolerance (pLI) scores for genes harboring heterozygous truncation mutations []. […]

Pipeline specifications

Software tools NovoAlign, Picard, GATK, CADD
Applications WGS analysis, WES analysis
Organisms Homo sapiens
Diseases Cardiomyopathy, Dilated, Heart Failure