Investigating the Faecal Microbiome in Formalin Fixed Paraffin Embedded (FFPE) Material

Purpose: Research into the faecal microbiome has shown a diverse population with a high level of variability between individuals. Altered faecal microbiomes are present in a range of diseases but work remains to understand their role in gastrointestinal disease. Current research into the microbiome makes use of fresh or frozen faecal samples. This restricts researchers to predominantly prospective study designs. One potential method for rapidly increasing and diversifying research is the retrospective study of FFPE material. We aimed to investigate the feasibility of typing the microbiome in FFPE faecal samples using next generation sequencing (NGS) technology. Methods: Material from six faecal samples was divided and stored as frozen or fixed and paran embedded creating two matched sub-groups. To assess assay sensitivity one sample was diluted to eight different concentrations before fixing and embedding. The V4 and V6 regions of the 16s rRNA gene were amplified. Primer pairs created approximately 240bp and 98bp targets in E.coli respectively. PCR products were multiplexed and sequenced on an Illumina MiSeq. QIIME software was used for analysis.Results: Analysis of alpha (within sample) diversity showed a signicant difference between sub-groups when targeting V4 (p=0.05) but not V6. Analysis of beta (between sample) diversity showed a significant difference between sub-groups when targeting V4 (p=0.01) while the V6 region showed a reduced, but still significant (p=0.02) dierence. The sensitivity assay showed comparable results down to 0.5% concentration levels. Conclusion: To our knowledge this is the first feasibility study generating NGS data on the microbiome from FFPE faecal material. Variation between matched frozen and FFPE faecal material was less when targeting V6 compared to V4. We hypothesise this may be due to the shorter amplicon undergoing less DNA fragmentation in FFPE material.