Long-read sequencing for reliably calling the <i>mompS</i> allele in <i>Legionella pneumophila</i> sequence-based typing

Sequence-based typing (SBT) of <i>Legionella pneumophila</i> is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires' disease. In the <i>L. pneumophila</i> SBT scheme, <i>mompS2</i> is one of seven genes that determine the sequence type (ST). The <i>Legionella</i> genome typically contains two copies of <i>mompS (mompS1</i> and <i>mompS2).</i> When they are non-identical it can be challenging to determine the <i>mompS2</i> allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 <i>L. pneumophila</i> genomes, there were 62 STs, 18 of which carried non-identical <i>mompS</i> copies. Using short-reads, the <i>mompS2</i> allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried <i>mompS1</i> allele 7 and <i>mompS2</i> allele 15, were assigned an incorrect <i>mompS2</i> allele and/or <i>mompS</i> gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical <i>mompS</i> copies, short-read assemblers occasionally failed to resolve the structure of the <i>mompS-</i>region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the <i>mompS</i>2 allele from short-reads, assess if other short-read methods were able to resolve the <i>mompS</i>-region, and investigate the possibility of using long-reads to obtain the <i>mompS</i> alleles, and thereby perform <i>L. pneumophila</i> SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the <i>mompS</i>-region and thus SBT from short-reads, but no method consistently solved the <i>mompS2</i> 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 <i>mompS</i> copies and correctly identify the <i>mompS2</i> 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 <i>L. pneumophila</i> SBT to overcome the <i>mompS</i> challenge observed with short-reads. To facilitate this, we have developed ONTmompS (https://github.com/marithetland/ONTmompS), an <i>in silico</i> approach to determine <i>L. pneumophila</i> ST from long-read or hybrid assemblies.