Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.
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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BibTeX
@misc{zmuda2026comprehensive,
title = {{Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.}},
author = {E. J. Zmuda and K. R. Covington and C. Kandoth and C. K. Y. Ng and D. J. Weisenberger and R. Bowlby and A. Chu and R. Corbett and D. Brooks and A. D. Cherniack and B. Murray and S. Ling and W. Zhao and E. Shinbrot and R. S. Lim and M. S. Bootwalla and T. Hinoue and J. Hu and N. Kakkar and M. Lawrence and M. McLellan and C. Miller and D. Morton and A. J. Mungall and D. Muzny and C. Sougnez and P. Stojanov and L. Xi and N. Schultz and R. Akbani and C. Curtis and B. Fulton and R. A. Gibbs and G. Getz and P. Spellman and M. Marra and M. Ladanyi and R. Tarnuzzer and H. Sofia and The Cancer Genome Atlas Research Network and C. Hutter and D. A. Wheeler and J. Gastier-Foster and Lair},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.31.748145},
url = {https://www.biorxiv.org/content/10.64898/2026.08.31.748145v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Comprehensive characterization of genomic, transcriptomic and epigenomic artifacts introduced in formalin-fixed, paraffin-embedded tissues.
AU - E. J. Zmuda
AU - K. R. Covington
AU - C. Kandoth
AU - C. K. Y. Ng
AU - D. J. Weisenberger
AU - R. Bowlby
AU - A. Chu
AU - R. Corbett
AU - D. Brooks
AU - A. D. Cherniack
AU - B. Murray
AU - S. Ling
AU - W. Zhao
AU - E. Shinbrot
AU - R. S. Lim
AU - M. S. Bootwalla
AU - T. Hinoue
AU - J. Hu
AU - N. Kakkar
AU - M. Lawrence
AU - M. McLellan
AU - C. Miller
AU - D. Morton
AU - A. J. Mungall
AU - D. Muzny
AU - C. Sougnez
AU - P. Stojanov
AU - L. Xi
AU - N. Schultz
AU - R. Akbani
AU - C. Curtis
AU - B. Fulton
AU - R. A. Gibbs
AU - G. Getz
AU - P. Spellman
AU - M. Marra
AU - M. Ladanyi
AU - R. Tarnuzzer
AU - H. Sofia
AU - The Cancer Genome Atlas Research Network
AU - C. Hutter
AU - D. A. Wheeler
AU - J. Gastier-Foster
AU - Lair
PY - 2026
DA - 2026/08/31
PB - bioRxiv
DO - 10.64898/2026.08.31.748145
UR - https://www.biorxiv.org/content/10.64898/2026.08.31.748145v1
AB - 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.
ER -