title: canine_ITS
design: Prospective study comparing cytological samples, aerobic bacterial cultures and culture-independent sequencing-based analyses of the external ear canal. Subjects with OE included 10 dogs with only cocci [≥25/high power field (HPF)] on cytological evaluation and culture of Staphylococcus spp.. 10 dogs with rods (≥25/HPF) and exclusive culture of Pseudomonas aeruginosa. 10 dogs with only yeast on cytological results morphologically compatible with Malassezia spp. (≥5/HPF).
library_construction_protocol: DNA was isolated from swab specimens as previously described. The ITS1F (CTTGGTCATTTAGAGGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC) primers were used for PCR amplification, each having a linker sequence, a sample specific GoLay12 index, and an Illumina adapter to amplify the ITS1 region of the fungal rRNA region. These indexed primers were used in combinations that made it possible to multiplex up to 576 (24 x 24) samples at a time. Each sample was amplified in duplicate, combined, and cleaned using the Agencourt AMPure XP bead-based PCR purification system (Beckman-Coulter). Purified amplicon pools were quantified using the Quant-IT dsDNA High-Sensitivity Assay Kit (Invitrogen) and a microplate reader (Thermo Scientific). A composite sample for sequencing was made by combining equimolar ratios of amplicons from the samples, followed by gel purification with a Qiagen MinElute Gel Extraction Kit to remove potential contaminants and PCR artifacts (the acceptable size window for amplicons was 200-1000 bp in length). The pooled DNA was quantified using a Qubit Fluorometer (Life Technologies), and PhiX174 genomic DNA was spiked in to the sample at ~40% prior to sequencing.