Lipworth, S;
Pickford, H;
Sanderson, N;
Chau, KK;
Kavanagh, J;
Barker, L;
Vaughan, A;
... Walker, A; + view all
(2020)
Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies.
Microbial Genomics
, 6
(11)
10.1099/mgen.0.000453.
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Abstract
Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in antimicrobial resistance/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data have suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore [Oxford Nanopore Technologies (ONT)] flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 h of sequencing, most chromosomes and plasmids had circularized and there was no benefit associated with longer running times. Quality was similar at 12 h, suggesting that shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilizing shorter run times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per-isolate sequencing cost. Ultimately this will facilitate large-scale studies utilizing hybrid assembly, advancing our understanding of the genomics of key human pathogens.
| Type: | Article |
|---|---|
| Title: | Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1099/mgen.0.000453 |
| Publisher version: | https://doi.org/10.1099/mgen.0.000453 |
| Language: | English |
| Additional information: | This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | bacterial genomics , Enterobacteriaceae , hybrid assembly , long-read assembly and Nanopore sequencing |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Inst of Clinical Trials and Methodology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > Inst of Clinical Trials and Methodology > MRC Clinical Trials Unit at UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10111199 |
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