16S rRNA Gene Sequencing Protocol

DNA was extracted from 200 μL of specimens on an automated MagNA Pure 96 isolation and purification system using the DNA and Viral NA small volume kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. DNA was eluted in a final volume of 100 μL, followed by a quantitative β globin assay to assess specimen adequacy, as previously described[36 (link)]. A quantitative 16S PCR was performed to report total bacterial load (16S rRNA gene copies per 5 μl of extracted DNA) using the broad range primer pair fD1 mod and 16S1RR-B, with 515F modified as a Taqman probe[37 (link)]. Specimens with insufficient DNA for amplification were re-extracted using an alternate methodology (S1 File). Twenty negative control samples were included to facilitate identification of reagent contaminants (S1 Table). Dual indexed universal primers Bakt_341F (CCTACGGGNGGCWGCAG) and Bakt_805R (GACTACHVGGGTATCTAATCC)[38 , 39 (link)] were used for PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene, as previously described[40 (link)]. Specimens and controls were sequenced on the Illumina MiSeq platform (Micromon, Monash University, Victoria, Australia).