title: Targeted pyrosequencing of 18S ribosomal RNA from sugar maple CWD decay class 3 : Sample MIS3.18s.1
design: DNA was extracted from coarse woody debris samples using the PowerPlant® DNA Isolation kit (MO BIO Laboratories Inc., Carlsbad, CA). A 5 minute homogenization step was performed using a 16 tube MiniBeadbeater TM (Biospec Products Inc., Bartlesville, OK) as opposed to the MO BIO vortex adapter described in the protocol. Samples were frozen and stored for analysis at a later date. Pyrotag sequencing of amplicons on the Roche 454 platform was completed on samples collected in 2013. All replicates of a given harvesting regime, herbicide use and type of coarse woody debris were pooled and sequenced at the Molecular Research LLP facility (Shallowater, TX, U.S.A.) per Dowd et al. (2008). Sequencing was done with Eubacterial forward primer (AGRGTTTGATCMTGGCTCAG).A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) were used under the following conditions: 94oC for 3 minutes, followed by 28 cycles of 94oC for 30 seconds;  53oC for 40 seconds and 72oC for 1 minute; after which a final elongation step at 72oC for 5 minutes was performed.  Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA).   Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents and following manufacturer’s guidelines sff dereplication was performed using QIIME.