Ion pgm template ot2 400 kit

The V4 variable region of the 16S rRNA gene was amplified using the primers 515F
(5′-GTGCCAGCM GCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′)¹¹. These primers were designed to include the adaptor sequences used in
the Ion Torrent NGS library preparation protocol containing the barcode sequence
on the forward primer. The samples were normalized to 12.5 ng/μL of DNA material
per library, and the amplification was performed in Veriti 96-well PCR equipment
(ThermoFisher, Waltham, Massachusetts, USA) followed by AMPure XP bead cleanup
(Beckman Coulter Life Sciences, Brea, California, USA). PCR conditions were 94
°C for 3 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing
at 58 °C for 30 s and extension at 68 °C for 1 min. PCR products were analyzed
by 1.5% agarose gel electrophoresis, stained with ethidium bromide solution and
visualized under ultraviolet light. Emulsion PCR was carried out using an Ion
PGM™ Template OT2 400 Kit (ThermoFisher, Waltham, Massachusetts, USA) in
accordance with the manufacturer’s instructions. Sequencing of the amplified
products was carried out using the Ion PGM™ Sequencing 400 Kit in an Ion Torrent
^TM Personal Genome Machine (ThermoFisher, Waltham, Massachusetts,
USA) using an Ion 318^TM chip kit v2 (ThermoFisher, Waltham,
Massachusetts, USA) with 16 libraries per chip. All samples were sequenced
once.