SugarShift Clinical Study, Havana, Cuba
16S amplicon sequencing was performed by EzBiome (Gaithersburg, MD, USA). Genomic DNA was quantified using a Qubit fluorometer (ThermoFisher, USA). The 16S rRNA V3-V4 regions were amplified using Illumina primers (F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG; R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) with gene-specific and Illumina adapter sequences. Amplicon PCR included 12.5 ng DNA, 12.5 L KAPA HiFi HotStart Ready Mix (Kapa Biosystems, Wilmington, MA), and 1 M primers in a 25 L reaction. Cycling conditions: 95C for 3 min, 25 cycles of 95C for 30 s, 55C for 30 s, 72C for 30 s, and a final 5 min at 72C. Products were cleaned with Mag-Bind RxnPure Plus magnetic beads (Omega Biotek, GA, USA). A second PCR for barcode and adapter incorporation used the same conditions but included 8 cycles. Libraries were normalized, pooled, checked on an Agilent 2200 TapeStation, and sequenced on a MiSeq (Illumina, San Diego, CA) with a 600-cycle (2  300 bp paired-end) run.