16S of human fecal content
fecal samples, collected from HAMSAB and placebo fed untreated human hypertensive subjects. Samples were sequenced for diversity profiling to compare the microbiome between the two groups (placebo) and (HAMSAB).  Polymerase chain reaction (PCR) amplification and sequencing were performed by the Australian Genome Research Facility (Melbourne, VIC).  PCR amplicons were generated using the 341F and 806R primers to amplify the V3-V4 region of the 16S gene. Thermocycling was completed with an Applied Biosystem 384 Veriti and using Platinum SuperFi mastermix (Life Technologies, Australia) for the primary PCR. The first stage PCR was cleaned using magnetic beads, and samples were visualised on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index the amplicons was performed with TaKaRa Taq DNA Polymerase (Clontech). The resulting amplicons were cleaned again using magnetic beads, quantified by fluorometry (Promega Quantifluor) and normalised.  The eqimolar pool was cleaned a final time using magnetic beads to concentrate the pool and then measured using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was diluted to 5 nM and molarity was confirmed again using a High-Sensitivity D1000 Tape. This was followed by sequencing on an Illumina MiSeq (San Diego, CA, USA) with a V3, 600 cycle kit (2 x 300 base pairs paired-end).