PCR of pond water community: Day 5 of transfer 2 - transferred to glucose, transferred back to cellulose (B)
Carried out by MRDNA Molecular Research, LP (Shallowwater, TX): The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer were used in a 28 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, multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads and used to prepare illumina DNA library. Sequencing was performed on a MiSeq following the manufacturers guidelines. Sequence data were processed using the MR DNA analysis pipeline. 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 fromRDPII and NCBI (www.ncbi.nlm.nih.gov, http://rdp.cme.msu.edu).