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 comprehen - sive 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 disadvan- tages. This pilot study compared four different library construction methods (Ion AmpliSeq, Illumina TruSeq, and Raindance Thunderbolts amplicon-based methods and Roche EZSeq se- quence capture method) and two different sequencing instruments (Ion Torrent PGM and Il- lumina 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 bioin- formatic pipeline for automated sequence data analysis was developed using the Galaxy en- vironment. 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.