Microbiome DNA extraction and 16S rRNA gene sequencing

Genomic DNA was extracted from all samples at Vanderbilt University Medical Center using the Qiagen DNeasy PowerSoil HTP Kit (96-well plates) following the manufacturer’s protocol, except the optional 4 °C incubations were skipped. Stool samples were thawed on ice and added directly to the kit plate. Nose, ear, and skin swabs were vortexed in tubes with 800 μL Qiagen PowerBead solution for 5 mins; this PowerBead solution was then added to the kit plate. An extraction negative, which did not contain any template but was otherwise processed the same as the rest of the samples, was included on each extraction plate. To mechanically lyse the cells, plates were shaken at 20 Hz in a TissueLyser II system (Qiagen) for 20 min. Steps 16–33 of the kit manufacturer’s protocol were performed on a QIAcube HT (Qiagen). One-step PCR targeting the V4 region of the 16S rRNA gene was performed using 515F/806R primers [75]. MyTaq HS Mix (Bioline) was used to create amplicons, with the following cycling conditions: 95 °C for 2 min; 30 cycles of 95 °C for 20 s, 50 °C for 15 s, 72 °C for 5 min; 72 °C for 10 min; 4 °C indefinitely. Positive PCR results were confirmed by the presence of a 400 bp band in 1% agarose gel electrophoresis; all negative controls were verified at this step to not have a visible band. The PCR products were cleaned and normalized using the SequalPrep Normalization Kit (Invitrogen). Samples and complementary controls (extraction negative, PCR negative, and ZymoBIOMICS Microbial Community Standard) were pooled and then cleaned using 1X AMPure XP beads. Sequencing was done on an Illumina MiSeq platform with 2x250bp reads at the Vanderbilt Technologies for Applied Genomics (VANTAGE) core facility.