Genome Med. 2015 Jul 16;7:71. doi: 10.1186/s13073-015-0197-4. eCollection 2015.

Achieving high-sensitivity for clinical applications using augmented exome sequencing.

Patwardhan A¹, Harris J¹, Leng N¹, Bartha G¹, Church DM¹, Luo S¹, Haudenschild C¹, Pratt M¹, Zook J², Salit M², Tirch J¹, Morra M¹, Chervitz S¹, Li M¹, Clark M¹, Garcia S¹, Chandratillake G¹, Kirk S¹, Ashley E³, Snyder M⁴, Altman R⁵, Bustamante C⁶, Butte AJ⁷, West J¹, Chen R¹.

Author information

1: Personalis, Inc, 1330 O'Brien Drive, Menlo Park, California 94025 USA.
2: Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, Maryland USA.
3: Personalis, Inc, 1330 O'Brien Drive, Menlo Park, California 94025 USA ; Center for Inherited Cardiovascular Disease, Stanford University School of Medicine, Stanford, California 94305 USA.
4: Personalis, Inc, 1330 O'Brien Drive, Menlo Park, California 94025 USA ; Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 USA.
5: Personalis, Inc, 1330 O'Brien Drive, Menlo Park, California 94025 USA ; Departments of Bioengineering & Genetics, Stanford University, Stanford, California 94305 USA.
6: Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 USA.
7: Personalis, Inc, 1330 O'Brien Drive, Menlo Park, California 94025 USA ; Institute for Computational Health Sciences, University of California, San Francisco, California 94158 USA.

Abstract

BACKGROUND:

Whole 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.

METHODS:

Using 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.

RESULTS:

We 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.

CONCLUSIONS:

Clinicians 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.