Two slices of formalin-fixed, paraffin-embedded (FFPE) tissue samples (about 10 mg of tissue) were used for total genomic DNA extraction. Samples were pre-treated at 55 °C for a minimum of 1 h with the dissolving buffer and Proteinase K (20 mg/ml) according to the manufacturer’s instructions (BiOstic® FFPE Tissue DNA Isolation Kit Mobio, Carlsbad, CA, USA). DNA was used to study the microbial diversity by pyrosequencing of the amplified V1–V3 region of the 16S rRNA gene, recurring to the primers Gray28F (5’-TTTGATCNTGGCTCAG) and Gray519r (5’-GTNTTACNGCGGCKGCTG) that amplify a fragment of 520 bp, following PCR conditions previously reported [17 (link)]. PCR products were purified twice with Agencourt AMPure purification kit (Beckman Coulter, Milan, Italy), and quantified using the PlateReader AF2200 (Eppendorf, Hamburg, Germany) with PicoGreen assay and an equimolar pool was obtained prior to further processing. Due to poor DNA quality, PG_8 sample was excluded. The amplicon pool was used for pyrosequencing on a GS Junior platform (454 Life Sciences, Roche, Monza, Italy) according to the manufacturer’s instructions by using Titanium chemistry.
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