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.