Freshwater monitoring by nanopore sequencing

Lara Urban; Andre Holzer; J Jotautas Baronas; Michael Hall; Philipp Braeuninger-Weimer; Michael J Scherm; Daniel J Kunz; Surangi N Perera; Daniel E Martin-Herranz; Edward T Tipper; Susannah J Salter; Maximilian R Stammnitz

We also found significant variations in relative abundances of the Leptospira genus, which was recently described to be enriched in wastewater effluents in Germany 27 . Indeed, the peak of River Cam Leptospira reads falls into an area of increased sewage influx (Figure 3c ). The Leptospira genus contains several potentially pathogenic species capable of causing life-threatening leptospirosis through waterborne infections 28 , however also features closerelated saprophytic and 'intermediate' taxa 29 . To resolve its complex phylogeny in the River Cam surface, we aligned Leptospira reads from all samples together with various reference sequences assigned to pre-classified pathogenic, saprophytic and other environmental Leptospira species 29 (Figure 3d ; Supplementary Table 4a; Methods). Despite the presence of nanopore sequencing errors (Extended Data Figure 8 ) and correspondingly inflated read divergence, we could pinpoint spatial clusters and a distinctly higher similarity between our amplicons and saprophytic rather than pathogenic Leptospira species. These findings were subsequently validated by targeted, Leptospira species-specific qPCR (Supplementary Table 5 , Methods), confirming that the latest nanopore sequencing quality is high enough to yield indicative results for bacterial monitoring workflows at the species level.