corn silage
Equimolar concentrations of PCR amplicons were pooled prior to paired-end sequencing (2  300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). The raw data were quality-filtered using Trimmomatic and merged with FLASH (Mago and Salzberg, 2011) based upon the criteria that follow: (i) all sequences were truncated at any ste that had a < 20 average quality score over a sliding 50 bp window; (ii) any reads were removed if they contained ambiguous bases or were exact matches to primer sequences with up to a two nucleotide mismatch; and (iii) any sequences that had > 10 bp of overlap were merged together. UPARSE (version 7.1) was used for operational taxonomic unit (OTU) clustering based upon a 97% similarity threshold, with UCHIME (Edgar, 2013) used for chimeric sequence identification and removal. To analyze the taxonomy, the Silva (SSU123) 16S rRNA database was used for alignment of 16S rRNA sequences based upon the RDP classifier algorithm (version 2.2) (Wang et al., 2007) with a 70% confidence threshold. Alpha diversity indices (Shannon index, Chao richness estimator, and Goods coverage estimator) and beta diversity indices were determined using QIIME (v1.7.0). R (v2.15.3) was used for principle component analysis (PCA).