The use of Oxford Nanopore native barcoding for complete genome assembly.
The Oxford Nanopore Technologies MinION(TM) is a mobile DNA sequencer that can produce long read sequences with a short turn-around time. Here we report the first demonstration of single contig genome assembly using Oxford Nanopore native barcoding when applied to a multiplexed library of 12 samples and combined with existing Illumina short read data. This paves the way for the closure of multiple bacterial genomes from a single MinION(TM) sequencing run, given the availability of existing short read data. The strain we used, MHO_001, represents the important community-acquired methicillin-resistant Staphylococcus aureus lineage USA300.



Using a hybrid assembly of existing short read and barcoded long read sequences from multiplexed data, we completed a genome of the S. aureus USA300 strain MHO_001. The long read data represented only ∼5% to 10% of an average MinION(TM) run (∼7x genomic coverage), but, using standard tools, this was sufficient to complete the circular chromosome of S. aureus strain MHO_001 (2.86 Mb) and two complete plasmids (27 Kb and 3 Kb). Minor differences were noted when compared to USA300 reference genome, USA300_FPR3757, including the translocation, loss, and gain of mobile genetic elements.



Here we demonstrate that MinION(TM) reads, multiplexed using native barcoding, can be used in combination with short read data to fully complete a bacterial genome. The ability to complete multiple genomes, for which short read data is already available, from a single MinION(TM) run is set to impact our understanding of accessory genome content, plasmid diversity, and genome rearrangements.
Sion C
SC
Bayliss
Vicky L
VL
Hunt
Maho
M
Yokoyama
Harry A
HA
Thorpe
Edward J
EJ
Feil
D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D015698:Genomic Library; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification / Q000235:genetics; D058608:Nanopores; D015203:Reproducibility of Results; D013045:Species Specificity
D004269:DNA, Bacterial
MinION; Staphylococcus aureus; Whole genome sequencing; bacterial genomics; hybrid assembly; long read; multiplexing; native barcoding