16S of Homo Sapiens fecal microbiome
DNA extraction was performed using a Qiagen DNeasy PowerSoil Pro DNA extraction kit (Qiagen, Venlo, The Netherlands). Up to 2 ml of the FIT solution was transferred to a new tube, and 200 L of 1 M Tris-HCl, pH 7.5, was added to quench the formaldehyde present in the FIT solution. After centrifugation, the supernatant was discarded, and the pellet resuspended in CD1 solution was transferred to PowerSoil Pro tubes. To increase the DNA yield, decrosslinking was performed by 4 hours of incubation at 65C with Proteinase K before the cell disruption step. The rest of the protocol was performed following the manufacturers instructions. DNA was quantified in all samples on a Qubit Fluorometer using a dsDNA HS Assay Kit (Thermo Fisher Scientific) and was then diluted to 5 ng/L for sequencing. Genomic DNA was amplified using the primers 16S_F (5- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG -3) and 16S_R (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC -3) for the PCR amplification of an approximately 460 bp region within the hypervariable (V3-V4) region of the prokaryotic 16S ribosomal RNA gene. Amplicon libraries for Illumina (Illumina, San Diego, USA) next-generation sequencing were generated by two-step PCR. First, a region specific for 16S rRNA was amplified via 24 cycles, and Illumina adapter and index sequences were then added via 7 cycles of PCR. The quality control of the amplicon libraries was performed via Agilent 2200 TapeStation analysis (Agilent Technologies, Santa Clara, USA) and with a Kapa Library Quantification Kit (Kapa Biosystems, Woburn, USA). The amplicon libraries were pooled in equimolar concentrations. Sequencing was carried out on an Illumina MiSeq System using a MiSeq Reagent Kit v3 in paired end 2300 bp mode.