While the fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to the DNA within the tissues. The DNA damage signature of FFPE DNA includes:
Hydrolysis of N-glycosyl bond
- ~ 0.5 depurination events yr-1 kb-1 (depyrimidation occurs at 5% of this rate). Rate significantly enhanced by lower pH.
- Mostly cytosine to uracil (~ 1 deamination yr-1 kb-1 for ssDNA and ~0.005 yr-1 kb-1 for dsDNA).
- 8-hydroxyguanine formation
- Thymine or cytosine glycol formation
- Thymine dimers
- Double strand breaks
Gathering meaningful biochemical data, including high-quality DNA sequence information, from these samples reproducibly poses a challenge. An additional challenge to analysis of archived tissue samples is that the typical sample is generally small and may only contain trace amounts of the tissue of interest and, therefore, the nucleic acid of interest. For these reasons, studies based on sequencing of FFPE samples have been technically challenging. Below is an example of a sample block of formalin-fixed, paraffin-embedded tissue.
- Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
- Protocol for use with NEBNext FFPE DNA Repair Mix (M6630) and NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)
- NEBNext FFPE DNA Repair Mix (M6630) - Protocol for use with Other User-supplied Library Construction Reagents
- High Quality Automation Libraries Prepared from a Broad Range of DNA Input Amounts (2017)
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Behind the paper: DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification
NEB researchers published a paper in Science highlighting DNA damage as a prevalent source of errors in public cancer databases. Learn about how addressing this damage could improve the detection of low-frequency disease variants.