Performance of non‐formalin fixed paraffin embedded samples in hybrid capture and amplicon next‐generation sequencing panels

Abstract

AbstractBackgroundGenomic profiling using next‐generation sequencing (NGS) is fundamental for driving prognostic and therapy in cancer. Formalin‐fixed paraffin embedded (FFPE) tissue is the widely used material, whereas non‐FFPE may represent an alternative. However, studies comparing the NGS performance of non‐FFPE materials to FFPE are still lacking in the literature. The objective of this study was to characterize in non‐FFPE preparations the nucleic acid yield and NGS performance on both a capture‐based and an amplicon‐based NGS platform. NGS quality metrics obtained from non‐FFPE preparations were compared to FFPE.MethodsWe analyzed the cellularity and nucleic acid yield in 111 tumors from non‐FFPE preparations. In addition, comprehensive hybrid capture panel sequencing metrics obtained from DNA and RNA libraries were compared between independent non‐FFPE and FFPE samples. A paired comparison between non‐FFPE and FFPE samples was performed to analyze concordance in mutant allele detection using an amplicon panel.ResultsThe mean target coverage from DNA libraries was 2× higher in non‐FFPE samples than in FFPE. The detection of exogenous DNA was 2.5× higher in non‐FFPE than in FFPE. Conversely, a lower performance was observed in non‐FFPE RNA libraries in comparison to FFPE DNA libraries with no impact in minimum standard cutoffs. The variant allele detection in non‐FFPE was found to be comparable to that of FFPE tumor samples in matched samples.ConclusionsNon‐FFPE was demonstrated to be a suitable material for DNA and RNA library preparations using a comprehensive NGS panel. This is the first study reporting library quality metrics according to the TSO500 analysis pipeline.