Matthew McCabe - Complete genome sequencing on the MinION corrects misidentification of Mannheimia varigena isolated from bovine milk by a veterinary diagnostic laboratory
Nanopore Community Meeting 2017
Mannheimia varigena is a gram negative bacillus that is pathogenic in ruminants. It has been isolated from pneumonic lungs (Harhay et al. 2014), udder and spleen (Blackall et al. 2002) of cattle. It has also been isolated from bovine meninges and was identified as the cause of fatal bovine meningitis (Catry et al. 2004). However, little is known about this pathogen and its ability to cause disease. So far, there are complete closed genome sequences available for only four M. varigena isolates which were all isolated from lung necroscopies taken from cattle with shipping fever and sequenced using the PacBio SMRT platform (Harhay et al. 2014). In the current study we used the MinION to sequence a bovine milk isolate that was identified by a veterinary diagnostic laboratory as Mannheimia haemolytica in March 2017 using conventional diagnostic methods (e.g. culture conditions, colony morphology, qPCR). For this we generated a sequencing library directly (without fragmentation) from 1 µg of extracted bacterial DNA using the 1D² Sequencing Kit (R9.5). The library was run on a FLO-MIN107 SpotON flowcell on a MinION Mk1B using MinKNOW (v1.7.14) software.
Sequencing was allowed to progress until approximately 200,000 sequence reads were generated. FASTQ files were assembled into a single contiguous sequence of 2.18 Mbp. NCBI nr/nt BLAST searches of protein sequences from predicted gene regions of the assembled genome showed full-length alignments of 99% -100% identity to all four Mannheimia varigena complete genome sequences (2.13-2.2 Mbp) and only 86% -87% identity to several Mannheimia haemolytica complete genome sequences (approximately 2.6-2.75 Mb in size). It is possible that Mannheimia varigina has been misidentified as Mannheimia haemolytica in veterinary diagnostic laboratories for a number of years resulting in an underestimation of the prevalence of Mannheimia varigina in ruminant pathology samples. This study shows that whole bacterial genome sequencing on the MinION is a single, facile analytical test which gives diagnostic accuracy that is unequivocal and superior to the current multiple, conventional, often subjective tests employed in diagnostic microbiology laboratories. Blackall PJ, Bisgaard M, Stephens CP. 2002. Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia varigena. Aust Vet J. 80:87-91.