Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Precision medicine relies on identifying actionable tumor mutations.
• Formalin-fixed paraffin-embedded (FFPE) tissues present challenges for accurate mutation detection due to DNA fragmentation and lesions.
• Sequence artifacts in FFPE DNA can be mistaken for true mutations, especially with tumor heterogeneity and next-generation sequencing.

Purpose of the Study:

• To review DNA template lesions in FFPE tissues as a source of sequence artifacts in molecular analysis.
• To discuss strategies for minimizing sequence artifacts and distinguishing them from true mutations.
• To provide guidance applicable to both single amplicon and massively parallel sequencing.

Main Methods:

• Review of literature on DNA lesions in FFPE tissues.
• Analysis of fragmentation, base modification (e.g., cytosine deamination), and abasic sites as sources of artifacts.
• Discussion of strategies to mitigate and identify sequence artifacts.

Main Results:

• Identified DNA fragmentation, base modifications (uracil, thymine from deamination), and abasic sites as key sources of sequence artifacts in FFPE DNA.
• Outlined strategies to minimize artifact generation and differentiate true mutations from artifacts.
• Demonstrated applicability of strategies for various sequencing approaches.

Conclusions:

• Maximizing accurate information from FFPE DNA is crucial as it's often the only available material.
• Minimizing sequence artifacts requires careful workflow management.
• Validation of actionable mutations using orthogonal methods or appropriate experimental design is recommended.