Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

Zmuda, E. J.; Covington, K. R.; Kandoth, C.; Ng, C. K. Y.; Weisenberger, D. J.; Bowlby, R.; Chu, A.; Corbett, R.; Brooks, D.; Cherniack, A. D.; Murray, B.; Ling, S.; Zhao, W.; Shinbrot, E.; Lim, R. S.; Bootwalla, M. S.; Hinoue, T.; Hu, J.; Kakkar, N.; Lawrence, M.; McLellan, M.; Miller, C.; Morton, D.; Mungall, A. J.; Muzny, D.; Sougnez, C.; Stojanov, P.; Xi, L.; Schultz, N.; Akbani, R.; Curtis, C.; Fulton, B.; Gibbs, R. A.; Getz, G.; Spellman, P.; Marra, M.; Ladanyi, M.; Tarnuzzer, R.; Sofia, H.; The Cancer Genome Atlas Research Network,; Hutter, C.; Wheeler, D. A.; Gastier-Foster, J.; Lair

doi:10.64898/2026.08.31.748145

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bioRxiv · 10.64898/2026.08.31.748145

Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.

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Abstract

Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.

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Zmuda, E. J., Covington, K. R., Kandoth, C., Ng, C. K. Y., Weisenberger, D. J., Bowlby, R., Chu, A., Corbett, R., Brooks, D., Cherniack, A. D., Murray, B., Ling, S., Zhao, W., Shinbrot, E., Lim, R. S., Bootwalla, M. S., Hinoue, T., Hu, J., Kakkar, N., Lawrence, M., McLellan, M., Miller, C., Morton, D., Mungall, A. J., Muzny, D., Sougnez, C., Stojanov, P., Xi, L., Schultz, N., Akbani, R., Curtis, C., Fulton, B., Gibbs, R. A., Getz, G., Spellman, P., Marra, M., Ladanyi, M., Tarnuzzer, R., Sofia, H., The Cancer Genome Atlas Research Network,, Hutter, C., Wheeler, D. A., Gastier-Foster, J., Lair. 2026-09-03. Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.. https://doi.org/10.64898/2026.08.31.748145

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