PCR amplicon of dog hookworms: ACH09
Total DNA was subjected to independent real-time PCR amplifications at the University of Sydney. ITS-2 rDNA (ITS-2) was amplified by the NC-1 and NC-2 primers, and two isoform-1 beta-tubulin regions were amplified using assay BZ167 and include key diagnostic SNPs conferring benzimidazole resistance. All primers were adapted for Illumina metabarcoding using Next Generation Sequencing (NGS). All PCR reactions were prepared using a Myra robotic liquid handling system (Bio Molecular Systems, Australia). Reactions were run on a CFX96 Real-Time PCR Detection System with corresponding CFX Manager software (BioRad, Australia). Amplicons were submitted for amplicon sequencing using Illumina NGS at the Ramaciotti Centre for Genomics, University of New South Wales, Sydney, Australia and sequenced on MiSeq (PE250) and MiSeq i100 PLUS (PE300).