16S rRNA gene seq DNA extraction contamination control
Breast milk samples were collected from mothers at the same time points as the fecal sampling from infants. Two different fractions of milk, namely foremilk and hindmilk was collected in participants home into disposable tubes using a manual breast pump (Type HarmonyTM, Medela AG, Baar, Switzerland) and stored at -20C until transportation (on ice) to the university where the samples were stored at -80 oC until DNA extraction. Prior to DNA extraction approximately 2 mL milk was centrifuged at 20.000 x g for 2 min. in micro-centrifuge tubes, after which the upper fat layer was removed using a pipette tip and the supernatant discarded by inverting the tube. The pellets were then re-suspended in 750 L bead solution (from the DNeasy PowerLyzer PowerSoil kit) and transferred to a new tube. Total DNA was extracted in random order from these suspensions 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. Blank DNA extraction controls (one for each batch of DNA extraction) were included following all the same steps as in the procedure for the milk samples. DNA concentrations were measured by Qubit dsDNA HS assay (InvitrogenTM, Q32851). PCR amplification and Ion Torrent sequencing was performed on DNA extracted from milk samples. Briefly, the V3-region of the 16S rRNA gene was amplified using 1-5 ng community DNA as template, 0.08 l AccuPrime Taq DNA Polymerase, high fidelity (Fisher Scientific: 12346086) , 2 l 10X Accuprime PCR Buffer II, 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 2 min at 94C, followed by 40 cycles of 94C for 20s and 58C for 20s and 68C for 20s and lastly 68C 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. A mock community (HM-276D, obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project) consisting of 200,000 16S rRNA genes embedded into the genomes of 20 bacterial species was used as template in triplicates for each intended sequencing run to control for inter and intra sequencing run consistency. 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 one chip together with blank DNA extraction controls and mock community using the Ion OneTouchTM and Ion PGM systems with a 318-Chip v2 incorporating the Hi-Q chemistry in 200 bp runs.