Abstract
BackgroundBacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools.ResultsPublicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids.ConclusionsWeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.
Highlights
Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance
Bacterial plasmids may contain additional genes, including genes involved in virulence, plasmid mobility and antibiotic resistance, which may confer the bacterium with fitness advantages under certain conditions, e.g., during treatment with antibiotics
We have developed WeFaceNano, an easy ‘to client’ application that is accessible via a web browser GUI that incorporates two fast long read sequence assemblers, quality control reporting, Basic Local Alignment Search Tool (BLAST) [12] database search function and an option to identify antibiotic resistance genes and plasmid incompatibility groups
Summary
Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. Dependent on the bacterial species, the size of the bacterial chromosomal ranges from 130 kbp [1] to over 14, 000 kbp [2], whereas plasmids tend to be relatively much smaller with a size that varies between 1 kbp to more than 200 kbp [3] These plasmids can be present as a single copy or in multiple copies within a single bacterium and replicate independently from the hosts’ chromosomal DNA. The detection, identification and tracking of plasmids within bacteria may allow interventions to be developed that lessen the impact of bacterial disease within human populations
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