Achieving high-sensitivity for clinical applications using augmented exome sequencing

Abstract

BackgroundWhole exome sequencing is increasingly used for the clinical evaluation of genetic disease, yet the variation of coverage and sensitivity over medically relevant parts of the genome remains poorly understood. Several sequencing-based assays continue to provide coverage that is inadequate for clinical assessment.MethodsUsing sequence data obtained from the NA12878 reference sample and pre-defined lists of medically-relevant protein-coding and noncoding sequences, we compared the breadth and depth of coverage obtained among four commercial exome capture platforms and whole genome sequencing. In addition, we evaluated the performance of an augmented exome strategy, ACE, that extends coverage in medically relevant regions and enhances coverage in areas that are challenging to sequence. Leveraging reference call-sets, we also examined the effects of improved coverage on variant detection sensitivity.ResultsWe observed coverage shortfalls with each of the conventional exome-capture and whole-genome platforms across several medically interpretable genes. These gaps included areas of the genome required for reporting recently established secondary findings (ACMG) and known disease-associated loci. The augmented exome strategy recovered many of these gaps, resulting in improved coverage in these areas. At clinically-relevant coverage levels (100 % bases covered at ≥20×), ACE improved coverage among genes in the medically interpretable genome (>90 % covered relative to 10-78 % with other platforms), the set of ACMG secondary finding genes (91 % covered relative to 4-75 % with other platforms) and a subset of variants known to be associated with human disease (99 % covered relative to 52-95 % with other platforms). Improved coverage translated into improvements in sensitivity, with ACE variant detection sensitivities (>97.5 % SNVs, >92.5 % InDels) exceeding that observed with conventional whole-exome and whole-genome platforms.ConclusionsClinicians should consider analytical performance when making clinical assessments, given that even a few missed variants can lead to reporting false negative results. An augmented exome strategy provides a level of coverage not achievable with other platforms, thus addressing concerns regarding the lack of sensitivity in clinically important regions. In clinical applications where comprehensive coverage of medically interpretable areas of the genome requires higher localized sequencing depth, an augmented exome approach offers both cost and performance advantages over other sequencing-based tests.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0197-4) contains supplementary material, which is available to authorized users.

Advancements in Next-Generation Sequencing

“…In contrast to the study by Patwardhan et al (90), other recent studies have found whole-genome sequencing to be superior to whole-exome sequencing in terms of overall variant sensitivity and a lack of bias because there is no selection procedure for whole-genome libraries. Lelieveld et al (66) found that whole-exome libraries required two to three times more coverage to achieve similar variant sensitivities compared with whole-genome libraries.…”

“…Patwardhan et al (90) evaluated the differential performance of four commercially available exome capture reagents and compared them with an augmented exome strategy that enhanced coverage over medically relevant genes. The authors reported superior variant sensitivities in the enhanced regions compared with traditional exome sequencing or whole-genome sequencing.…”

The Use of Next-Generation Sequencing for Research and Diagnostics for Intellectual Disability

“…The American College of Medical Genetics and Genomics has issued the professional standards and guidelines for implementation of NGS in clinical laboratories (Rehm et al 2013). In the light of these guidelines, Patwardhan et al (2015) have evaluated the performance of the commercially available exome-capture methods and sequencing technologies. Insufficient reads coverage was observed across several disease-associated genes using each of the conventional exome capture and whole-genome platforms.…”

Precision Medicine in Gastrointestinal Pathology

“…57 A survey of commonly used methods that cover greater than 90% of targeted nucleotides demonstrated that complete data were returned from these methods on only 30% to 65% of genes of medical relevance. 58 In addition to this potential lack of complete analysis of genes of interest, there is a requirement for a minimal number of reads at each position of interest. Unfortunately, many MPS tests are often described in terms of median or mean coverage.…”