16S rRNA analysis of thermophilic anaerobic switchgrass digester: reduced retention times
DNA isolation with PowerLyzer Powersoil DNA Isolation kit and Precellys 24 high-throughput tissue homogenizer. DNA eluted in 100 ?l of water and assessed by Nanodrop and 1% agarose gel. 16S rDNA amplicon pool was prepared according to the Lundberg et al. 2013 method (17) with 5 cycles of amplification: denaturing the reaction  at 95 °C for 1 min, annealing at 50 °C for 2 min, extension at 72 °C for 2 min and cooled down to 4°C. The product was purified with 17 ?l of Agencourt Ampure XP beads and eluted in 21 ?l of water. The primers for tagging were a mixture of 515 F and 806R for 16S rDNA V4 region. For the second PCR step, 20 ?l of purified DNA was used in a 50 ?l reaction. The PCR program was 1 cycle at 95°C for 45 sec and 32 cycles at 95°C for 15 sec, annealing at 60°C for 30 sec, and 72°C for 30 sec. For sequencing, the DNA samples were pooled together, purified with Agencourt Ampure XP beads, PhiX diluted, and denatured following the manufacturers protocol.  The final sequencing concentration was 9 pM with 80% amplicon pool and 20% PhiX.  Paired end sequencing (2 x 251 bp) was completed on an Illumina MiSeq instrument with a v2 500 cycle kit.