title: Abundance and short-term temporal variability of fecal microbiota in healthy dogs
design: Assessment of fecal microbiota  Extraction of DNA. Genomic DNA was extracted and purified from 100 mg of each fecal sample using a phenol-chloroform extraction procedure as described by Suchodolski et al. (2005).    454-pyrosequencing. The fecal bacterial taxonomic structure was evaluated using a bacterial tag-encoded FLX-titanium 16S rRNA gene amplicon pyrosequencing (bTEFAP) as described previously for canine fecal samples (Handl, et al., 2011). The sequence processing pipeline was carried out as described in detail by Garcia-Mazcorro et al. (2012). Briefly, Phred20 quality reads were trimmed to remove tags and primer sequences, depleted of non-16S rRNA reads, chimeric, mitochondrial, and sequences of less than 250 bp length. The final sequences were evaluated using BLASTN against a continually curated high-quality 16S rRNA gene database derived from the National Center for Biotechnology Information (NCBI). BLAST outputs were compiled to generate percentage files at each taxonomic level. Sequences with identity scores to known of well-characterized 16S rRNA gene sequences between 95% and 97% identity were resolved at the genus level, between 90% and 95% at the family level, between 85% and 90% at the order level, between 80% and 85% at the class level, and 77-80% at the phylum level. Sequence information is available through GenBank within a short read archive (SRA) under accession (?) .