16S rRNA Gene Amplification and Sequencing

Bacterial DNA was extracted as described previously [43 (link)] using the PureLink kit (K182002, Invitrogen) and quantified using the Quant-iT PicoGreen Assay Kit (P7589, Invitrogen). The 16S ribosomal RNA genes were amplified using bar-coded PCR primers targeted to the V1-V3 hypervariable region (FP 5′-AGAGTTTGATCCTGGCTCAG-3′; RC 5-ATTACCGCGGCTGCTGG-3′). PCR reactions were carried out in quadruplicate using Accuprime Taq HiFi (12346086, Invitrogen). Each PCR reaction contained 0.2 µM of each primer, 1 U Accuprime Taq HiFi, 1× Buffer II, and 2 µL DNA in a total volume of 25 µL. Cycling conditions are as follows: 1 cycle of 95 °C for 2 min; 32 cycles of 95 °C for 20 s, 60 °C for 30 s, and 72 °C for 60 s; 1 cycle of 72 °C for 5 min. The resulting 16S rDNA amplicons were purified using a 1:1 volume of SPRI beads (09-981-123, GE Healthcare, Chicago, IL, USA), quantified using PicoGreen, pooled in equal amounts, and sequenced on the Illumina MiSeq using 2 × 300 bp chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to assess potential environmental contamination. Library preparation and sequencing were performed at the CHOP Microbiome Center (University of Pennsylvania, Philadelphia, PA, USA).