Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing

Abstract

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.

Fig. 1. Sequencing and assembly statistics for the Zymo mock bacterial species (n = 7).

a, Observed raw read accuracies measured through read-mapping. b, Observed homopolymer length of raw reads compared with the reference genomes (see Supplementary Figs. 2 and 3 for a complete overview). c, Observed indels of de novo assemblies per 100 kbp at different coverage levels, with and without Illumina polishing. Note that the reference genomes available for the Zymo mock are not identical to the sequenced strains (Supplementary Table 3). d, IDEEL score, calculated as the proportion of predicted proteins that are ≥95% the length of their best-matching known protein in a database. The dotted line represents the IDEEL score for the reference genome, while the dashed lines mark a 40-fold coverage cut-off.