title: Mudpot Metagenome (Consolation Prize Mudpot)
design: The 16S rRNA gene V4 variable region PCR primers 515/806 (OR OTHER PRIMER SELECTED) with barcode on the forward primer  were used in a 30 cycle PCR  (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed.  After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples are pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines. Sequence data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). In summary, sequences were joined, depleted of barcodes then sequences <150bp removed, sequences with ambiguous base calls removed. Sequences were denoised, OTUs generated and chimeras removed. Operational taxonomic units (OTUs)were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated database derived from GreenGenes, RDPII and NCBI (www.ncbi.nlm.nih.gov,  DeSantis et al 2006, http://rdp.cme.msu.edu).