16S of Homo Sapiens fecal microbiome
For bacterial DNA isolation, first the samples were homogenized in a Stomacher-400 blender. QIAamp Stool Mini Kit (Qiagen, Venlo, The Netherlands) was used for DNA extraction. The standard protocol was followed, with the exception that the samples were mixed with the lysis buffer and incubated at 95oC instead of 70oC in order to ensure the lysis of both Gram-negative and Gram-positive bacteria. The extracted DNA was quantified with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, DE, USA). DNA yield was determined by measuring absorbance ratios spectrophotometrically, and included A260/280 nm for protein and A260/230 nm for salt and phenol contamination. Microbiome was profiled by sequencing the V3-V4 regions of the 16S rRNA gene on an Illumina MiSeq instrument with forward 5-CCTACGGGNGGCWGCA-3 and reverse 5- GACTACHVGGGTATCTAATCC-3 primers. All PCR reactions were performed in 25 l volume containing 12.5 l 2X KAPA HiFi Hotstart ready mix (KAPA Biosystems, Woburn, MA, USA), 5 L of each primer (1M), and 2.5 L of extracted DNA (10 ng) under the following cycling conditions: initial denaturation at 95oC for 3 min, followed by 35 cycles of denaturation at 95oC for 30 s, annealing at 55oC for 30 s, and elongation at 72oC for 30 s, with a final extension at 72oC for 5 min. PCR clean-up was done with AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA). Next, a PCR to index the amplicons was performed using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) with conditions: 95oC for 3 min; 8 cycles of 95oC for 30 s, 55oC for 30 s, 72oC for 30 s, with a final extension step of 5 min at 72oC, and hold at 4oC. Next, the pooled PCR products were purified using AMPure XP beads (Beckman Coulter) before quantification. The final library was paired-end sequenced (2 x 300 bp) using a MiSeq Reagent Kit v.3 on the Illumina MiSeq sequencing system (Illumina). Raw sequences were demultiplexed with Illumina bcl2fastq2 Conversion Software v2.20 and raw data were imported into open-source software QIIME 2 2019.7 (36) using the q2-tools-import script with PairedEndFastqManifestPhred33 input format. In total, 16,063,617 (average 78,743 per person) reads were generated with the V3-V4 16S rRNA sequencing. Denoising was done with DADA2 (37), using a quality-aware model of Illumina amplicon errors to attain an abundance distribution of sequence variances, which have a difference of a single nucleotide. After retrieving quality scores, q2-dada2-denoise script was used to truncate the forward reads at position 288 and trim at position 16. Reverse reads were truncated at position 241, trimming was not applied on the reverse read. Chimera removal was done using the consensus filter via q2-dada2-denoise in which chimeras are detected in samples individually and sequences found chimeric in a sufficient fraction of samples are removed. During this step forward and reverse reads were also merged.