DNA extraction, PCR amplification and sequencing
All samples were subjected to genomic DNA extraction, followed by 16S ribosomal RNA based PCR amplification to allow for bacterial DNA sequencing and analysis. Genomic DNA was extracted from swabs using a LEV blood kit (Promega, Madison, WI) implemented on a Maxwell 16 instrument, following manufacturer’s protocol with several modifications. Modifications included a lysozyme incubation (10 ng/µL lysozyme; Thermo Fisher Scientific) for 30 min at 37°C, followed by bead-beating (40 s at 6 m/s) using a FastPrep-24 System (MP Biomedicals). Homogenized samples were transferred to the Maxwell cartridges for final purification of DNA.
Genomic DNA was prepared for sequencing using a two-stage amplicon sequencing workflow, as described previously (19). Initially, genomic DNA was PCR amplified using primers targeting the V4 region of microbial 16S ribosomal RNA (rRNA) genes. The primers, 515F modified and 806R modified (20), contained 5’ linker sequences compatible with Access Array primers for Illumina sequencers (Fluidigm, South San Francisco, CA). PCRs were performed in a total volume of 10 microliters using MyTaq™ HS 2X Mix (Bioline), primers at 500 nM concentration and approximately 1,000 copies per reaction of a synthetic double-stranded DNA template (described below). Thermocycling conditions were as follows: 95°C for 5’ (initial denaturation), followed by 28 cycles of 95°C for 30 sec, 55° for 45 sec, and 72°C for 30 sec. One microliter of PCR product from each reaction was transferred to the second-stage PCR reaction. Each reaction was conducted in a final volume of 10 microliters using MyTaq HS 2X mix, and each well contained a unique primer pair of Access Array primers containing Illumina sequencing adapters, single index sample-specific barcode, and linker sequences. Thermocycling conditions were as follows: 95°C for 5’ (initial denaturation), followed by 8 cycles of 95°C for 30 sec, 60° for 30 sec, and 72°C for 30 sec. Libraries were pooled and purified using 0.6X concentration of AMPure XP beads to remove short fragments below 300 bp.