Amplicon sequencing of Cyp51 from a fungal pathogen using unique molecular identifiers

This study used the Oxford Nanopore MinION portable DNA sequencer to sequence the Cyp51 gene from two closely related fungal pathogens, Pyrenophora teres f. teres and Pyrenophora teres f. maculata using Unique Molecular Identifiers (UMIs). The pipeline was validated using genomic DNA from fungal isolates with known Cyp51A haplotypes (Cyp51A mock community) and then applied to cultured field samples. Clusters generated from the pipeline delivered SNP-level accuracy for single base pair mutations, however some discernibly low density clusters contained errors in homopolymer regions. This method can be easily expanded to include other fungicide target genes and also be used to profile mutations in other systems where chemical resistance is an issue such as human pathogens, insects and weeds.