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Title: Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization.
Austin Authors: Do, Hongdo;Dobrovic, Alexander 
Affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia
School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia
Issue Date: 24-Nov-2014
Publication information: Clinical Chemistry 2014; 61(1): 64-71
Abstract: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations.This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches.Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.
Gov't Doc #: 25421801
DOI: 10.1373/clinchem.2014.223040
Type: Journal Article
Subjects: Artifacts
DNA Damage
False Negative Reactions
False Positive Reactions
Paraffin Embedding
Reproducibility of Results
Sequence Analysis, DNA
Templates, Genetic
Tissue Fixation
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