bacterial community of horse manure- 1WHM
Samples (~10 g) of aging horse manure (from fresh to 5 weeks old) used in the oviposition assays were collected weekly (on the day of the two-choice experiment) and frozen in -80°C for later analysis. Total genomic DNA was extracted from the fecal samples (0.5 g) using FastDNA® SPIN kit (MP Biomedicals) following manufacturer’s instructions. The bacterial tag-encoded FLX Titanium 16S rDNA amplicon parallel pyrosequencing and post sequencing processing were carried out at the Medical Biofilm Research Institute (Lubbock, TX) as described by Dowd et al. (2008) and Middelbos et al. (2010). Briefly, primers extended from 27F numbered in relation to the Escherichia coli 16S ribosomal gene were used. A single step reaction with 30 cycles was used and 0.5 U of HotStar HiFidelity Polymerase (Qiagen Inc, Valencia, CA) was added to each reaction. Labeling and pyrosequencing was conducted as described in Dowd et al. (2008). Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Sequence collections were depleted of chimeras using B2C2 software. The resulting files were then depleted of short reads (<350 bp), reads with ambiguous base calls, reads with homopolymers >6 bp, and reads with lower than Q25 quality scores. Data were analyzed and interpreted using Sequencher 4.8 (Gene Codes) for alignment and sequence editing, MOTHUR (Schloss et al., 2009) for diversity and richness, and Blast2GO (www.blast2go.com) for the NCBI GenBank search.