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Updated: Jul 7, 2025
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Performance of non-formalin fixed paraffin embedded samples in hybrid capture and amplicon next-generation sequencing
Sibele Inácio Meireles1, Mariana Vargas Cruz1, Carla Daniele de Godoy1
1Departamento de Anatomia Patológica e Molecular, Hospital Sírio Libanês, São Paulo, Brazil.
Background:
Genomic 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.
Methods:
We 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.
Results:
The 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.
Conclusions:
Non-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.
