16S rRNA amplicons (RT-PCR)
Reverse-transcriptase-PCR of the partial 16S rRNA was performed using the universal primers F343 (5’- TACGGRAGGCAGCAG-3’) and R533 (5’- ATTACCGCGGCTGCTGGC-3’) containing the 10-bp multiplex identifiers (MID) and adaptor sequences for Ion Torrent sequencing described previously (1, 2). Reactions were performed using a Qiagen OneStep RT-PCR kit (QiaGen, Valencia, CA) in 25-μl volumes containing 2 μl of template RNA (50-100 ng/µl), 5µl of 5X reaction buffer, 0.6 μM of each primer, 0.4 mM of dNTPs and 1 μl of enzyme mix. Cycling conditions involved a reverse-transcriptase step at 50°C for 30 min, an initial 15 min denaturing step at 95°C, followed by 15 cycles of 30 s at 94°C, 30 s at 55°C, and 60 s at 72°C, and a final elongation step of 10 min at 72°C. All PCR products were purified on agarose gels using the QIAquick Gel Extraction Kit (Qiagen) and quantified using the PicoGreen dsDNA quantitation assay (Invitrogen, Carlsbad, CA). All amplification reactions from the 24 different samples were pooled in an equimolar ratio and sequenced together. A total of 3.50x107 molecules were used in an emulsion PCR using the Ion OneTouch 200 Template Kit v2 (Life Technologies) and the OneTouch instruments (Life Technologies) according to the manufacturer’s protocol.