16S rRNA-Seq of MSM: NA149
There were 200 available urine samples from study participants. These samples were refrigerated between 2-8C until processed. A total of 10 ml of each urine was centrifuged and the supernatant discarded. The recovered pellets were then subjected to further molecular analyses. DNA was extracted from the sample pellets using available Microbiome Kit and quantified using the Nanodrop Spectrophotometer. The extracted DNA was stored at 20C until subsequent analysis.The V3  V4 hypervariable regions of the 16S ribosomal ribonucleic acid (rRNA) gene from urine genomic DNA (gDNA) were amplified using forward primer 5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG- 3 and reverse primer 5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3. Reactions were performed in a total volume of 25 L. The DNA library was prepared using the PCR products, following the Illumina TruSeq Nano DNA Sample Preparation protocol. Sequencing was performed on an Illumina MiSeq, using a 2 x 301 cycle paired-end approach.