High throughput exome coverage of clinically relevant cardiac genes

Abstract

BackgroundGiven the growing use of whole-exome sequencing (WES) for clinical diagnostics of complex human disorders, we evaluated coverage of clinically relevant cardiac genes on WES and factors influencing uniformity and depth of coverage of exonic regions.MethodsTwo hundred and thirteen human DNA samples were exome sequenced via Illumina HiSeq using different versions of the Agilent SureSelect capture kit. 50 cardiac genes were further analyzed including 31 genes from the American College of Medical Genetics (ACMG) list for reporting of incidental findings and 19 genes associated with congenital heart disease for which clinical testing is available. Gene coordinates were obtained from two databases, CCDS and Known Gene and compared. Read depth for each region was extracted from the exomes and used to assess capture variability between kits for individual genes, and for overall coverage. GC content, gene size, and inter-sample variability were also tested as potential contributors to variability in gene coverage.ResultsAll versions of capture kits (designed based on Consensus coding sequence) included only 55% of known genomic regions for the cardiac genes. Although newer versions of each Agilent kit showed improvement in capture of CCDS regions to 99%, only 64% of Known Gene regions were captured even with newer capture kits. There was considerable variability in coverage of the cardiac genes. 10 of the 50 genes including 6 on the ACMG list had less than the optimal coverage of 30X. Within each gene, only 32 of the 50 genes had the majority of their bases covered at an interquartile range ≥30X. Heterogeneity in gene coverage was modestly associated with gene size and significantly associated with GC content.ConclusionsDespite improvement in overall coverage across the exome with newer capture kit versions and higher sequencing depths, only 50% of known genomic regions of clinical cardiac genes are targeted and individual gene coverage is non-uniform. This may contribute to a bias with greater attribution of disease causation to mutations in well-represented and well-covered genes. Improvements in WES technology are needed before widespread clinical application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-014-0067-8) contains supplementary material, which is available to authorized users.

Comprehensive Screening of Eight Known Causative Genes in Congenital Hypothyroidism With Gland-in-Situ

“…Additionally, although the study was not designed to allow direct comparison of different sequencing methods, the rate of causative mutations in cases screened using either the most sensitive technique (MiSeq targeted sequencing, in which exons with <20-fold coverage were individually resequenced using Sanger sequencing) or WES, was similar and supported our conclusion that approximately 40% cases are unsolved. Previous studies have also reported considerable variability in uniformity and depth of coverage across the exome, and these data, together with our sequencing depth analysis, highlight a limitation of targeted sequencing, which may impact and limit variant identification (33). High-depth, whole-genome sequencing can improve exon coverage and the advent of recent sequencing technologies (such as the Illumina X10 system) makes this possible at large scale.…”

“…CH severity is classified according to European Society for Paediatric Endocrinology criteria on the basis of serum fT4 levels; severe, <5, moderate 5 to <10, and mild >10 pmol/liter, respectively (33) and pathogenicity is predicted according to American College of Medical Genetics guidelines (34). A schematic of the TG protein illustrates the position of the mutations relative to the key structural domains of TG including the repetitive type 1, 2, and 3 cysteine-rich regions, acetylcholinesterase homology (ACHE-like) domain and hormonogenic domains.…”

“…CH severity is classified according to European Society for Paediatric Endocrinology criteria (33) and pathogenicity is predicted according to American College of Medical Genetics guidelines (34). Mutation position is illustrated using a schematic representation of the domain structure of the DUOX2 protein.…”

Aortic dilation, genetic testing, and associated diagnoses

“…15 With the known phenotypic overlap among CTDs and the sometimes imperative need to provide better counseling and guide clinical monitoring and intervention, NGS panels have been designed to explore simultaneously several genes known to cause different CTDs. 16,17 Including patients who had both NGS panels and conventional Sanger sequencing studies, molecular genetic studies for possible CTDs were performed in only 9% of patients. When those patients thought to have AoD caused by BAV or other conotruncal heart defects were excluded, the proportion of patients who underwent such studies was significantly higher.…”

Enhancing Literacy in Cardiovascular Genetics: A Scientific Statement From the American Heart Association

“…A recent study reported considerable variability in the coverage of known cardiac genes depending on the types of sequence capture kit and depth of sequencing used. 47 Sequence capture is the process of enrichment of selected genomic regions from whole genomic DNA and is required to capture the coding regions of the genome when exome sequencing is performed.…”