Application of Targeted Next-generation Sequencing, TruSeq Custom Amplicon Assay for Molecular Pathology Diagnostics on Formalin-fixed and Paraffin-embedded Samples.

Abstract

The implementation of targeted therapies revolutionized oncology. As the number of new oncogenic driver mutations, which provide molecular targets for prediction of effective and selective therapies, is increasing, the implementation of fast and reliable methods by molecular pathology labs is very important. Here we report our results with TruSeq Custom Amplicon assay performed on formalin-fixed and paraffin-embedded material. The oligo capture probes targeted the hotspot regions of 10 well-known oncogenes linked to clinical diagnosis and treatment of lung and colorectal adenocarcinomas, melanomas, and gastrointestinal stromal tumors. Fifteen previously genotyped formalin-fixed and paraffin-embedded DNA samples from different tumor types were selected for massively parallel sequencing. A bioinformatics pipeline was developed to identify high-quality variants and remove sequence artifacts. With the exception of 1 sample, which was of lower quality than the others, relevant mutations corresponding to tumor types could be reliable detected by the developed bioinformatical pipeline. This study indicates that the application of TruSeq Custom Amplicon assay is a promising tool in molecular pathology diagnostics, but it is important to standardize sample processing (including fixation, isolation procedure, sample selection based on quality assessment, and rigorous variant calling) to achieve the highest success rate and avoid false results.