Research Article: Exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Holly LaDuca, Kelly D. Farwell, Huy Vuong, Hsiao-Mei Lu, Wenbo Mu, Layla Shahmirzadi, Sha Tang, Jefferey Chen, Shruti Bhide, Elizabeth C. Chao, Noam Shomron.


With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference.

Corresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.

When assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.

Results from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.

Partial Text

With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians are faced with the decision to pursue targeted gene panels versus whole exome sequencing (WES) as their first-tier testing approach [1]. This decision may be particularly challenging for diseases with significant genetic and phenotypic heterogeneity. It is important to consider the benefits and limitations of each approach when deciding on the best testing strategy.

The internal database at Ambry Genetics (Aliso Viejo, CA) was queried for all alterations classified as pathogenic and likely pathogenic (herein referred to collectively as ‘pathogenic variants’) detected on germline targeted NGS multi-gene panel testing of over 50,000 patients in the clinical diagnostic laboratory from April 2010 until July 2014. Variants underwent thorough assessment and review of available evidence and were classified using a five-tiered classification algorithm [8] based on guidelines from the American College of Genetics and Genomics and the International Agency for Research on Cancer [9, 10]. Pathogenic variants in this study included single nucleotide substitutions and small insertion and deletion events up to 40 basepairs. Gross rearrangements, large insertions, and large deletions detected on array or multiplex ligation-dependent probe amplification-based assays were excluded from analysis. Multi-gene panels targeted a range of hereditary (Mendelian) disorders including cancer susceptibility, X-linked intellectual disability (XLID), primary ciliary dyskinesia (PCD), Marfan syndrome, thoracic aortic aneurysms and dissections, and related disorders (Marfan/TAAD), and other cardiovascular diseases such as cardiomyopathies and arrhythmias, and were selected by the ordering clinician based on the patients’ clinical history. Corresponding nucleotide positions for these pathogenic variants were interrogated against data from 100 randomly-selected patients whose samples were submitted to Ambry Genetics for clinical WES, and the coverage at each position was assessed. Pathogenic variants were interpreted as having adequate depth for detection if coverage at the respective nucleotide position on WES was ≥10X. Coverage at the first and last nucleotides was averaged for insertions, and for deletions and indels coverage was assessed for the first and last nucleotides, with the lower of the two values being used for analysis. Since all data was accessed anonymously, this research was determined to be exempt from review based on 45.CFR46.101 and is in compliance with the Helsinki Declaration (Solutions Institutional Review Board, Reference Number 1OCT14-93).

A total of 1533 different pathogenic variants identified on targeted NGS multi-gene panel testing were included in this analysis, representing 91 genes implicated in 5 disease categories identified through analysis of greater than ~100,000 alleles (S1 Table). Pathogenic variants in cancer susceptibility genes accounted for 88.1% of pathogenic variants analyzed (n = 1350), with each of the other disease categories accounting for <4% of pathogenic variants studied. Exonic single nucleotide substitutions were the most common type of variant included in this analysis (n = 665, 43.4%), followed by small deletions (n = 485, 31.6%), intronic variants (n = 184, 12.0%), small duplications and insertions (n = 169, 11.0%), and indels (n = 30, 2.0%). Despite current estimates that 90–95% exome-wide coverage is achieved with WES, results from this position-specific comparative coverage analysis limited to disease-causing variants identified through NGS panels demonstrate that exome sequencing is expected to perform well (≥98.5%) for a range of inherited diseases. If validated in follow-up studies, these data will help guide clinicians in deciding which type of testing to pursue for their patients. These data suggest the use of exome sequencing may achieve similar diagnostic yield when compared to panel based tests and, if cost and turn-around-time are comparable or favorable, that WES may be an appropriate first-tier option to consider when clinically indicated. The high level coverage achieved by WES reported herein, coupled with high rate of newly characterized and novel gene findings on exome (30% collectively) [6] demonstrates a major benefit of WES compared to panel testing for Mendelian diseases.   Source:


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