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Updated: Feb 6, 2026

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples
Published on: June 8, 2020
Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests
David Millán-Esteban1, Diana Reyes-García2, Zaida García-Casado1
1Laboratorio de Biología Molecular, Instituto Valenciano de Oncología, Valencia, Spain.
Abstract:
The use of NGS in clinical practice for precision diagnosis requires a quality starting material. Despite the broadly established use of formalin-fixed paraffin-embedded (FFPE) samples in molecular testing, these usually have low-quality DNA. We established a method to determine the suitability of melanoma FFPE samples for an amplicon-based NGS custom panel analysis. DNA was extracted from unstained melanoma samples and wide local excision samples. Amplicon-based libraries were constructed and tested using time and quality parameters as variables. Time elapsed from sample retrieval >7 years, a quality control value > 5.63 and a DNA integrity value < 2.05 indicated samples were not suitable. A decision tree is provided with rate of samples suitable for analysis according to the combination of these parameters.
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