Optimized De novo assembly of Haemophilus influenzae using oxford nanopore sequencing without short-read data on a...

Oxford Nanopore sequencing enables rapid, long-read, and cost-effective bacterial genome analysis; however, historically high error rates and reliance on command-line interface (CLI)-based workflows have limited its accessibility in clinical laboratories. We optimized a fully graphical user interface (GUI)-based workflow using the open-source Galaxy platform (usegalaxy.eu) for de novo assembly of Haemophilus influenzae genomes using Nanopore data alone, without short-read correction. Sixteen strains were sequenced on a MinION Mk1B with R9.4.1 and R10.4.1 flow cells. Read trimming, filtering, assembly, and polishing were systematically evaluated within Galaxy. Assembly accuracy was assessed against hybrid assemblies using SNP and insertion/deletion (Indel) counts, multilocus sequence typing (MLST), penicillin-binding protein 3 (PBP3) mutation detection, and phylogenetic clustering. Optimal performance was achieved with R10.4.1 using Dorado super accurate (SUP) basecalling, ≥1 kb read filtering, quality score ≥10, Flye assembly, and Medaka polishing with the bacterial methylation model. Under these conditions, the error rate was 0.0007%, MLST and PBP3 mutation detection showed 100% concordance with hybrid assemblies, and phylogenetic clustering was fully consistent. In THUN1414 subsampling, 100 × coverage appeared sufficient to maintain this accuracy. In contrast, R9.4.1 yielded an error rate of 0.1989% without full concordance. R9.4.1 data contained more Indels, particularly in repetitive sequences, whereas R10.4.1 data exhibited a higher frequency of residual SNP differences; most remaining discrepancies were enriched in homopolymeric tracts or other low-complexity regions. This study provides a proof-of-concept, species-focused technical validation of a fully GUI-based Nanopore-only bacterial assembly workflow and highlights key considerations for broader application and future multicenter validation.