Characterizing the microbial community response to modulations in prairie-based anaerobic digestion parameters
16S rRNA gene amplicon sequencing was completed using a modified version of the Earth Microbiome Project 16S Illumina amplicon protocol [18]. Universal 16S rRNA gene primers targeting the V4 variable region 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806RB (5-GGACTACNVGGGTWTCTAAT-3) were used. Amplicons were generated using the Platinum Hot Start PCR Master Mix (2x) (ThermoFisher) and the following conditions: initial denaturation step at 94 C for 3 min; 45 s at 94 C; 60 s at 50 C; 90 s at 72 C. This was repeated for 35 cycles and finished with a 10 min extension at 72 C. The resulting amplicons were quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) and purified using the standard protocol of the QIAquick 96 PCR Purification Kit (Qiagen, Hilden, Germany). All described reagents were dispensed using a Mantis robot (Formulamatrix). The barcoded amplicons underwent paired-end 500-cycle sequencing on an Illumina MiSeq platform. Reads were demultiplexed by the facility prior to analysis.
SUB16178106
2_B06_Recirc_R4_2