Nanopore adaptive sampling effectively enriches bacterial plasmids

Abstract

Bacterial plasmids play a major role in the spread of antibiotic resistance genes. However, their characterization via DNA sequencing suffers from the small proportion of plasmid DNA in those samples. Although sample preparation methods can enrich the proportion of plasmid DNA before sequencing, these methods are expensive and laborious, and they might introduce a bias by enriching only for specific plasmid DNA sequences. Nanopore adaptive sampling could overcome these issues by rejecting uninteresting DNA molecules during the sequencing process. In this study, we assess the application of adaptive sampling for the enrichment of low abundant plasmids in known bacterial isolates using two different adaptive sampling tools. We further inspect potential cost savings for laboratories by utilizing adaptive sampling on reused or expired flow cells. We show that a significant enrichment can be achieved even on reused or expired flow cells. By applying adaptive sampling, we also improve the quality of de novo plasmid assemblies and reduce the sequencing time. Our experiments indicate that the choice of the read classification method and read lengths used for the classification influence the enrichment factor.

Targeted sequencing ofEnterobacteralesbacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies

“…Hybrid capture-based methods suffer from high financial costs and a lack of target flexibility (Hodges et al 2007; Brown et al 2015). ONT’s adaptive sampling shows promise for depleting unwanted DNA during sequencing, but has thus far been unable to substantially increase absolute numbers of target sequences and often leads to premature flowcell degradation (Loose et al 2016; Edwards et al 2019; Kovaka et al 2021; Payne et al 2021; Ulrich et al 2024).…”