16S rRNA Gene V4 Amplification

For universal amplification of the V4 region of the 16S rRNA gene (V4 iTags), we used forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and reverse primer 806R (5′-GGACTACHVGGGTTCTAAT-3′) containing a variable 12 bp barcode sequence [56 (link)]. Pooled amplicons were purified with the Agencourt AMPure XP purification system (Beckman Coulter, Brea, CA, USA) and analyzed with an Agilent bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) to confirm appropriate amplicon size. iTag sequencing was performed according to JGI’s standard procedures: iTag amplicons were diluted to 10 nM, quantified by quantitative PCR and sequenced on the Illumina MiSeq platform (reagent kit v.3; Illumina Inc., Carlsbad, CA, USA). iTag sequences were analyzed using the JGI iTagger analysis pipeline [57 (link)].