SSU amplicon / 454 from horse placenta
To identify the potential intracellular and free eukaryotes present in the diagnostic specimen, we used a community profiling based amplification 454 sequencing approach of conserved eukaryotic SSU rDNA. Genomic DNA (gDNA) was isolated from approximately 25 mg of frozen (-20°C) allantochorion using the Isolate II Genomic DNA kit (BioLine, Australia), according to manufacturer’s instructions. The gDNA was submitted to the diversity profiling service at the Australian Genomic Research Facility, Australia. The SSU rDNA assay applied was based on primer pair Euk1A (5’-CTGGTTGATCCTGCCAG-3’) and Euk516 (5’-CCAGACTTGCCCTCC-3’), amplifying approximately 500-550 bp of the 5’-end of the eukaryotic SSU rDNA. The barcoded PCR amplicon was pooled and sequenced on the 454 GS‐FLX platform.