Development of a multilocus sequence typing method for accurate identification of 16SrV phytoplasma strains via Oxford nanopore

The elm yellows phytoplasma group (16SrV) includes diverse members associated with devastating plant diseases on a broad host range across Europe, Asia and Americas. To enable the detection and precise identification of a number of 16SrV phytoplasma strains, it was developed and assessed a multilocus sequence analysis approach that combines targeted PCR pre-amplification of DNAs with nanopore sequencing. Three housekeeping genes (16S rRNA; secY; map) were amplified from each sample and sequenced simultaneously using a single-use Flongle Flow Cells. Furthermore, it was developed a bioinformatic analysis pipeline to run BLAST searches of assembled reads against a curated DNA sequence database of 16SrV phytoplasmas. Preliminary results on selected DNA samples suggest that this method can produce sufficient reads and consensus sequence information across the three selected markers to allow accurate determination of phytoplasma strains identity in less than two hours. Moreover, by barcoding the amplicons produced during library preparation, it was possible to process up to 24 different samples in one sequencing experiment therefore reducing the overall cost per sample. Owe to the improved R10.4.1 Flow Cell and the high sequencing depth, consensus sequences produced by this method were at least as accurate as those obtained by Sanger sequencing and allowed differentiation of closely related strains of group 16SrV and the identification of mixed infections.