16S of Homo Sapiens fecal microbiome
DNA extraction was performed using a Qiagen DNeasy PowerSoil Pro DNA extraction kit (Qiagen, Venlo, The Netherlands). ~200 mg of stool was used following the DNA extraction kit manufacturers instructions, except the samples were incubated for an additional 10 minutes at 65 C after adding solution CD1. Cell disruption step was performed using a Precellys 24 tissue homogenizer (parameters: 2x30 sec, 2500 rpm, 30-second pause). 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.