title: 16S rRNA amplicon sequencing of peat: MireWide.201507_Plot21_1_10 (alternate name: X21M)
design: From each sample, 0.25g were used for DNA extraction using the PowerSoil-htp 96 Well Soil DNA Isolation Kit (cat # 12955-4, Qiagen, Hilden, Germany) per the manufacturers protocol. Extracted DNA was quantified using Qubit 2.0 (Invitrogen, Waltham, United States) and stored at -80C until 16S rRNA amplicon sequencing at Argonne National Laboratory. There, genomic DNA was amplified using the Earth Microbiome Project barcoded primer set, adapted for the Illumina MiSeq by adding nine extra bases in the adapter region of the forward amplification primer that support paired-end sequencing (Caporaso 2010, Caporaso 2012). The V4 region of the 16S rRNA gene (515F-806R) was amplified with region-specific primers that included the Illumina flowcell adapter sequences.  The reverse amplification primer also contained a twelve base barcode sequence that supported pooling of up to 2,167 different samples in each lane. Each 25ul PCR reaction contained 12ul of MoBio PCR Water (Certified DNA-Free), 10ul of 5 Prime HotMasterMix (1x), 1ul of Forward Primer (5uM concentration, 200pM final), 1ul Golay Barcode Tagged Reverse Primer (5uM concentration, 200pM final), and 1ul of template DNA. The conditions for PCR were: 94C for 3 minutes, followed by 35 cycles at 94C for 45 s, 50C for 60 s, and 72C for 90 s; with a final extension of 10 min at 72C. The PCR amplicons were quantified using PicoGreen (Invitrogen) and a plate reader. Once quantified, different volumes of each of the products were pooled into a single tube so that each amplicon was represented equally. This pool was then cleaned up using UltraClean PCR Clean-Up Kit (Qiagen), and then quantified using the Qubit. After quantification, the molarity of the pool was determined and diluted down to 2nM, denatured, and then diluted to a final concentration of 4.0pM with a 10% PhiX spike for sequencing on the Illumina MiSeq (300 cycles).