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

To identify if DNA reads assigned to Leptospiraceae were more similar to sequence reads of previously identified pathogenic, intermediate or environmental Leptospira species, we built a neighbour-joining tree of Leptospiraceae reads classified in our samples data, together with sequences from reference databases (Figure 3d ; species names and NCBI accession numbers in clockwise rotation around the tree in Supplementary Table 4a ). We matched the orientation of our reads, and then aligned them with 68 Leptospira reference sequences and the Leptonema illini reference sequence (DSM 21528 strain 3055) as outgroup. We then built a neighbour-joining tree using Muscle v.3.8.31 67 (excluding three reads in the 'Other Environmental' clade that had extreme branch lengths >0.2). The reference sequences were annotated as pathogenic and saprophytic clades P1, P2, S1, S2 as recently described 29 .