16S rRNA gene seq infant feces
Fecal samples were collected from participants and stored at -80C until DNA extraction from 200 mg feces in random order using the DNeasy PowerLyzer PowerSoil kit (Qiagen, 12855-100) with the following modification: bead beating was performed at 30 cycles/s for 10 min (Retsch MM 300 mixer mill) and the initial centrifugation steps were performed at 10,000 x g for 3 min, as recommended for clay matter. DNA concentrations were measured by Qubit dsDNA HS assay (InvitrogenTM, Q32851). PCR amplification and Ion Torrent sequencing was performed on DNA extracted from fecal material. Briefly, the V3-region of the 16S rRNA gene was amplified using 1-5 ng community DNA as template, 0.2 l Phusion High-Fidelity DNA polymerase (Fisher Scientific, F-553L), 4 l HF-buffer, 0.4 l dNTP (10 mM of each base), 1 M forward primer (PBU 5-A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3) and 1 M reverse primer (PBR 5-trP1-adapter-ATTACCGCGGCTGCTGG-3) in 20 l total reaction volume. Both primers (TAG Copenhagen A/S) were linked to sequencing adaptors and the forward primer additionally contained a unique 10 bp barcode (Ion Xpress Barcode Adapters) for each sample. The PCR program consisted of initial denaturation for 30s at 98C, followed by 24 cycles of 98C for 15s and 72C for 30s, and lastly 72C for 5 min to allow final extension before cooling to 4C. No template controls were included for each PCR run, all resulting in less 0.05 ng/l. The PCR products were purified by use of HighPrepTM PCR Magnetic Beads (MAGBIO, AC-60005) with a 96-well magnet stand (MAGBIO, MyMag 96), according to the manufacturers recommendations. DNA quantity was measured using Qubit dsDNA HS assay (InvitrogenTM, Q32851) and samples were pooled to obtain equimolar libraries and sequenced on two separate chips using the Ion OneTouchTM and Ion PGM systems with a 318-Chip v2 incorporating the Hi-Q chemistry in 200 bp runs.