Characterization of FFPE-induced bacterial DNA damage and development of a repair method

Abstract

Formalin-fixed, paraffin-embedded (FFPE) specimens have huge potential as source material in the field of human microbiome research. However, the effects of FFPE processing on bacterial DNA remain uncharacterised. Any effects are relevant for microbiome studies, where DNA template is often minimal and sequences studied are not limited to one genome. As such, we aimed to both characterise this FFPE-induced bacterial DNA damage, and develop strategies to reduce and repair this damage. Our analyses indicate that bacterial FFPE DNA is highly fragmented, a poor template for PCR, crosslinked and bears sequence artefacts derived predominantly from oxidative DNA damage. Two strategies to reduce this damage were devised - an optimised decrosslinking procedure reducing sequence artefacts generated by high-temperature incubation, and secondly, an in vitro reconstitution of the base excision repair pathway. As evidenced by whole genome sequencing, treatment with these strategies significantly increased fragment length, reduced the appearance of sequence artefacts, and improved the sequencing readability of bacterial and mammalian FFPE DNA. This study provides a new understanding of the condition of bacterial DNA in FFPE specimens and how this impacts downstream analyses, in addition to a strategy to improve the sequencing quality of bacterial and possibly mammalian FFPE DNA.

The effect of the formalin-fixed paraffin-embedded process on salivary microbiota profiling

“…FFPE-based microbiota analysis in tissues is expected to lead to the elucidation of pathogenesis and the identification of future diagnostic and therapeutic targets. Formalin fixation during the FFPE process induces various types of damage to DNA, including fragmentation, artificial mutations of bases, and cross-linking (DNA-DNA, Protein-DNA) due to the action of formaldehyde (Williams et al, 1999;Srinivasan et al, 2002;Do and Dobrovic, 2015;Flores Bueso et al, 2020). The potential damage inflicted by formaldehyde on DNA templates can produce sequence artifacts that may cause erroneous results in microbiota analysis utilizing NGS.…”

Bladder cancer-associated microbiota: Recent advances and future perspectives

“…However, the tissues are not sterile during paraffin embedding, and DNA degradation may occur within the paraffin-embedded specimens. Therefore, the use of paraffinized tissues for high-throughput microbiome sequencing requires the elimination of the “noise” from paraffin [ 48 , 49 ]. Studies comparing FF and FFPE tissues have been performed in other cancer types [ 50 ].…”

Understanding and harnessing triple-negative breast cancer-related microbiota in oncology

“…The bacterial load is of extremely low biomass relative to the TME or the GM for that matter and issues of contamination and sampling techniques are important confounding factors. While our lab and others to improve the relevant methodology ( 153 – 156 ), bacteria at the scene cannot be pinned down to having a causative effect in oncogenesis or are merely bystanders having found a niche where they can survive ( 135 ).…”