Epmotion 5075 liquid handling workstation

For each sample, the V1V2 region of the bacterial 16S rRNA gene was amplified with Golay bar-coded universal primers 27F and 338R (Table S3) (41 (link), 46 (link), 47 (link)). PCRs were performed in triplicate with 5 µl DNA, 7.21 µl PCR-grade water, 2.5 µl 10× buffer II, 0.19 µl Taq, 5 µl of the forward primer (2 µM), and 5 µl of the reverse primer (2 µM). PCRs were prepared in a PCR clean room using an EpMotion 5075 liquid handling workstation (Eppendorf, Hamburg, Germany) and run in quadruplicate on an Applied Biosystems GeneAmp PCR 9700 system (Thermo Fisher Scientific Inc., Waltham, MA) under the following cycling conditions: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 90 s; and a final extension at 72°C for 8 min. Reaction replicates were pooled into libraries and purified using Agencourt AMPure XP beads (Beckman Coulter, Inc., Indianapolis, IN) per the manufacturer’s protocol. The purified PCR products were then pooled and sequenced using an Illumina MiSeq platform. qPCR quantification of 16S rRNA gene copies was used to ensure that a minimum of 1,000 16S rRNA gene copies were input into each reaction. To pass our quality filter, samples were also required to contain at least 1,000 Illumina sequence reads.

DNA was extracted from stool using the MagAttract PowerSoil DNA Kit on an Eppendorf epMotion 5075 Liquid Handling Workstation.16S rRNA sequencing was performed on all stool samples. For bacterial sequencing, we amplified the V3-V4 regions of the 16S rRNA gene using standard primers with Illumina Nextera adaptors (Illumina, Madison WI). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ) and quantified using the Quant-iT Broad Range dsDNA Assay kit (Thermo Fisher Scientific, Waltham, MA). Libraries were normalized and pooled with a 10% PhiX spike and sequenced on an Illumina MiSeq with a v3 kit. Negative controls were included on all sequencing runs.