Anne Vatland Kr��vel, Marit A K Hetland, Eva Bernhoff, Anna Steensen Bj��rheim, Markus Andr�� Soma, Iren H L��hr
Sequence-based typing (SBT) of is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires' disease. In the SBT scheme, is one of seven genes that determine the sequence type (ST). The genome typically contains two copies of and When they are non-identical it can be challenging to determine the allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 genomes, there were 62 STs, 18 of which carried non-identical copies. Using short-reads, the allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried allele 7 and allele 15, were assigned an incorrect allele and/or gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical copies, short-read assemblers occasionally failed to resolve the structure of the region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the 2 allele from short-reads, assess if other short-read methods were able to resolve the -region, and investigate the possibility of using long-reads to obtain the alleles, and thereby perform SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the -region and thus SBT from short-reads, but no method consistently solved the allele. By using Oxford Nanopore Technology (ONT) sequencing together with Trycycler and Medaka for long-read assembly and polishing we were able to resolve the copies and correctly identify the allele, in accordance with Sanger sequencing/EQA results for all tested isolates (n=35). The remaining six genes of the SBT profile could also be determined from the ONT-only reads. The STs called from ONT-only assemblies were also consistent with hybrid-assemblies of Illumina and ONT reads. We therefore propose ONT sequencing as an alternative method to perform SBT to overcome the challenge observed with short-reads. To facilitate this, we have developed ONTmompS (https://github.com/marithetland/ONTmompS), an approach to determine ST from long-read or hybrid assemblies.
