Comparison of Next-Generation Sequencing Platforms for Clinical Testing of Non-Small Cell Lung Cancer

Abstract

Personalized treatment of lung cancer using therapies that target activating oncogenic 
mutations such as EGFR and ALK has become the standard of care. Current molecular testing 
is routinely performed for single genes and increasingly in a multiplex format. However, the 
scarcity of sufficient biopsy material has necessitated a more high-throughput and comprehensive testing approach. Next Generation Sequencing (NGS) offers great promise as a highly 
sensitive method of detection for a variety of biopsy sources (tissue, blood, pleural effusions). 
However, there are multiple NGS platforms and panels with varying advantages and disadvantages. This pilot study compared four different library construction methods (Ion AmpliSeq, 
Illumina TruSeq, and Raindance Thunderbolts amplicon-based methods and Roche EZSeq sequence capture method) and two different sequencing instruments (Ion Torrent PGM and Illumina MiSeq). A common set of ten tumor/normal pairs from lung adenocarcinoma patients 
were analysed by all platforms. Additional samples were analysed in subsets of the platforms. 
To assess the feasibility of sequencing circulating free DNA (cfDNA) from plasma and pleural 
effusions, two additional samples were analysed on two amplicon-based platforms. A bioinformatic pipeline for automated sequence data analysis was developed using the Galaxy environment. To determine the most cost-effective, technically streamlined library construction 
and sequencing method, we compared coverage statistics, sensitivity, variant detection, and 
workflow for all platforms.