title: Amplicon-Seq of digester metagenome: M2 - 80C
design: Samples were collected from each digester effluent stream and stored at -20C until further analyses. Genomic DNAs from biomass samples were extracted using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). V4 16S rRNA gene amplification, PCR conditions, primer selection, and sequence library preparation were done using the Earth Microbiome Project protocols and standards (EMP 16S Illumina Amplicon Protocol) (Caporaso et al., 2012, Parada et al., 2016) using Thermo Fisher Platinum Taq DNA Polymerase. Sequencing libraries were run on the Illumina MiSeq platform using services provided by the University of British Columbia Sequencing and Bioinformatics Consortium. A custom pipeline leveraging USEARCH (Edgar, 2010) and QIIME (Caporaso et al., 2010) modules was applied in order to determine microbial community structure based on V4 16S rRNA gene sequences. UCHIME was used to identify potential chimeric sequences using the RDP gold database (Cole et al., 2009, Edgar et al., 2011). QIIMEs pick_otus.py was used to identify OTUs de novo at 97% sequence similarity from the chimera-filtered fragments using SUMACLUST. A representative sequence set was selected from the de novo OTUs by pick_rep_set.py prior to taxonomic assignment using the RDP classifier against QIIMEs default database. Chimeras and singletons were removed to avoid Type 1 errors ultimately resulting in the OTU table used for downstream analyses and data visualization.