Cross-Comparison of Exome Analysis, Next-Generation Sequencing of Amplicons, and the iPLEX(®) ADME PGx Panel for Pharmacogenomic Profiling.

Eng Wee Chua,Klaus Lehnert,Kim N T Ton,Phillip Shepherd,Martin A Kennedy,Nuala Helsby,Simone L Cree

doi:10.3389/fphar.2016.00001

Abstract

Whole-exome sequencing (WES) has been widely used for analysis of human genetic diseases, but its value for the pharmacogenomic profiling of individuals is not well studied. Initially, we performed an in-depth evaluation of the accuracy of WES variant calling in the pharmacogenes CYP2D6 and CYP2C19 by comparison with MiSeq® amplicon sequencing data (n = 36). This analysis revealed that the concordance rate between WES and MiSeq® was high, achieving 99.60% for variants that were called without exceeding the truth-sensitivity threshold (99%), defined during variant quality score recalibration (VQSR). Beyond this threshold, the proportion of discordant calls increased markedly. Subsequently, we expanded our findings beyond CYP2D6 and CYP2C19 to include more genes genotyped by the iPLEX® ADME PGx Panel in the subset of twelve samples. WES performed well, agreeing with the genotyping panel in approximately 99% of the selected pass-filter variant calls. Overall, our results have demonstrated WES to be a promising approach for pharmacogenomic profiling, with an estimated error rate of lower than 1%. Quality filters, particularly VQSR, are important for reducing the number of false variants. Future studies may benefit from examining the role of WES in the clinical setting for guiding drug therapy.

Highlights

Whole-exome sequencing (WES) is an increasingly important technology in rare-disease (Maxmen, 2011) and drug-response genetics (Price et al, 2012)
We initially compared the WES data quality for two key pharmacogenes, CYP2D6 and CYP2C19, with data generated by an amplicon sequencing assay we developed on the MiSeq R (Illumina) platform, for the same 36 samples (Supplementary Methods; per-exon depths of coverage are presented in Supplementary Figures S1 and S2)
For the 43 variant sites identified by WES a total of 943 individual WES genotype calls were generated across the 36 samples, but 202 calls were excluded for being of insufficient quality or because MiSeq R amplicon sequencing data were not available

Summary

Introduction

Whole-exome sequencing (WES) is an increasingly important technology in rare-disease (Maxmen, 2011) and drug-response genetics (Price et al, 2012). Besides being an effective tool for detecting potentially diseasecausing variant(s), WES can provide added information on variation in pharmacogenes. Though WES data has been shown to be highly accurate in previous studies, provided that appropriate quality filters are applied 2014), none of these studies have explored the use of WES data for pharmacogenomic profiling. The accuracy of WES variant calling could be compromised by the failure of the technology to resolve highly similar genes (Drögemöller et al, 2013). Sequencing of the CYP2D6 gene is confounded by the presence of closely related pseudogenes, CYP2D7 and CYP2D8, such that pre-amplification with long-range PCRs is usually applied to avoid undesired sequence contamination (Stüven et al, 1996)

Methods

Results

Conclusion