Optimized use of Oxford Nanopore flowcells for hybrid assemblies

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.

Keywords: Enterobacteriaceae; Nanopore sequencing; bacterial genomics; hybrid assembly; long-read assembly.

Fig. 1.

Schematic representation of experiments performed, flowcells used and libraries sequenced. *The same 12 isolates were sequenced in both libraries 3 and 4, but with different barcodes, as shown in the inset table and Table 1.

Fig. 2.

Number of contigs generated by Unicycler for the nine included barcoded (bc) samples in library 1 over time (one isolate was excluded, see the Methods section). Complete assemblies (where the chromosome and all plasmids are formed of single circularized contigs) are shown in blue.

Fig. 3.

Assembly likelihoods were calculated with the assembly likelihood estimator (ALE) by mapping Illumina reads to hybrid assemblies. Likelihoods were calculated for assemblies of each barcoded isolate (y-axis) created at 6 h intervals up to 48 h (x-axis). A likelihood difference of 0 (blue) implies that the assembly at time t is equally as likely as that at t=48 h. A positive likelihood difference (orange) implies that the assembly at time t was better than at 48 h and a negative likelihood difference (green) implies a worse assembly at time t vs 48 h.

Fig. 4.

Output over time for flowcell 3 (libraries 3/4/5, left) and flowcell 1 (library 1, right). The ONT wash kit was used between libraries 3/4/5 on flowcell 3, whereas flowcell 1 was run for 48 h with no washing steps.