Bouras, G.; Judd, L. M.; Edwards, R. A.; Vreugde, S.; Stinear, T. P.; Wick, R. R.

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing is still required for perfection. However, the effect of short-read depth on polishing performance is not well understood. Here, we introduce Pypolca (with default and careful parameters) and Polypolish v0.6.0 (with a new careful parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default and Polypolish-careful commonly introduce false-positive errors at low depth; (2) most of the benefit of short-read polishing occurs by 25x depth; (3) Polypolish-careful never introduces false-positive errors at any depth; and (4) Pypolca-careful is the single most effective polisher. Overall, we recommend the following polishing strategies: Polypolish-careful alone when depth is very low (<5x), Polypolish-careful and Pypolca-careful when depth is low (5-25x), and Polypolish-default and Pypolca-careful when depth is sufficient (>25x).