Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice

PloS One
Leah M PrenticeBrandon S Sheffield

Abstract

Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE) material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG) treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality <30. A larger baseline group of samples (n = 20) was examined to esta...Continue Reading

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Citations

Apr 12, 2019·PloS One·Samantha J McDonoughJulie M Cunningham
Nov 13, 2019·The American Journal of Surgical Pathology·Jennifer PorsLynn N Hoang
Jul 23, 2020·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·William Mathieson, Geraldine A Thomas
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Related Concepts

Deamination
False Positive Reactions
Formalin
Colorectal Neoplasms
Paraffin Embedding
Sequence Determinations, DNA
Computational Molecular Biology
Uracil-DNA glycosylase
High-Throughput Nucleotide Sequencing
Cytosine

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