Nanopore Sequencing of Bacterial Biothreat Agents

Here we present DNA isolation, rapid and field library preparation, and nanopore sequencing for Y. pestis and B. anthracis. Two commercially available DNA isolation kits were used to produce genomic DNA (gDNA) of sufficient quality and quantity for MinION WGS using both rapid and field library preparation methods. MinION reads are error-prone compared to Illumina and PacBio WGS data; containing more indels, particularly in homopolymer regions. When mutations are not located in homopolymeric regions, SNPs are accuratly called. Large and small plasmids are consistently assembled to completion from MinION data, with antimicrobial resistance genes encoded on these plamids readily identified. Expected and novel mulltimeric forms of the Y. pestis plasmid, pPCP1, were assembled, but these forms were not identified by Illumina or PacBio sequencing. A retrospective down sampling analysis revealed that 100,000 raw (.fast5) reads were sufficient for >99% coverage of B. anthracis and Y. pestis chromosomes and plasmids, as well as accurate detection of antimicrobial resistance markers and mutations. The ability sequence and analyze a Y. pestis or B. anthracis genome from a culture isolate in < 8.5 hours using nanopore sequencing could contribute to an expedited public health response.