Nanopore-only assembly and methylation profiling of Arabidopsis thaliana across chemistries, basecalling models and assembly workflows

Over the past decade, nanopore sequencing has progressed rapidly through advances in flow cell chemistry, library preparation, basecalling algorithms and base-modification detection. These developments have increased both throughput and read accuracy, making nanopore-only sequencing increasingly practical for eukaryotic genome assembly and direct detection of DNA methylation. Here, we present nanopore sequencing data, genome assemblies, and methylation analysis for the Columbia (Col-0) accession of the model plant Arabidopsis thaliana and assess how successive improvements in the nanopore sequencing platform have affected read accuracy and assembly quality. Data from earlier R9.4.1 and R10.4 flow cells were sufficient to generate nanopore-only multi-megabase and chromosome-arm scale assemblies, with higher reference-alignment identity achieved with the improved R10.4 chemistry. Super-accuracy rebasecalling increased read quality in both datasets and markedly improved assembly identity and gene completeness. Methylation calls obtained directly from nanopore signal were strongly concordant with a published bisulphite sequencing dataset at shared covered sites, with Pearson's r values of 0.93 and 0.96 for the R9.4.1 and R10.4 datasets, respectively. To assess current nanopore assembly performance, we additionally analysed 14.3 Gb R10.4.1 simplex reads, basecalled with a super-accuracy model and assembled using hifiasm, a contemporary long-read de novo assembler. This produced a five-contig nuclear assembly of 134.93 Mb, with each contig corresponding to one of the five Arabidopsis nuclear chromosomes. The assembly recovered 99.9% complete BUSCO genes and showed 99.98% one-to-one alignment identity to TAIR12, with three chromosomes assembled telomere-to-telomere. These results provide an empirical example of how nanopore-only plant genome assembly has improved across successive workflows and demonstrate the value of nanopore data for simultaneous genome assembly and methylation analysis.