Nanopore sequencing reaches amplicon sequence variant (ASV) resolution

Sequencing of ribosomal marker genes remains a cornerstone for profiling complex microbial communities. In recent years, there has been a shift from Illumina to long-read technologies, including PacBio and Oxford Nanopore Technologies (ONT). ONT is attractive due to its low startup cost and portability; however, historically high error rates have prevented direct amplicon sequencing variant (ASV) generation from raw nanopore reads. This has forced most workflows to rely on mapping raw reads against reference databases constraining analyses to taxa covered by these. With recent improvements in ONT sequencing accuracy, we sought to challenge this view by sequencing samples of increasing complexity using primer sets targeting amplicons of different lengths, and by sequencing the exact same PCR libraries on both PacBio and ONT. We demonstrate that error-free ASVs can now be generated directly from raw nanopore reads using standard denoising algorithms originally developed for Illumina data. Current ONT read quality enables reliable reconstruction of amplicons spanning [~]250 bp to [~]4,200 bp and allows resolution of intragenomic rRNA gene variants. These results extend beyond simple mock communities to complex fecal, anaerobic digester, activated sludge, and soil samples. When sequencing depth is sufficient, ONT accurately recovers all or nearly all intra-genomic 16S rRNA gene copy variants, showing perfect sequence identity to curated reference sequences in mock communities and to ASVs inferred from PacBio data in complex communities. Across the primer sets, ONT required higher sequencing depth than PacBio to fully resolve the communities, with this requirement increasing with amplicon length. For complex samples, ONT required approximately 2-3x more reads for V4 ([~]250 bp) and V1-V3 ([~]500 bp), 4.1-5.6x more reads for V1-V8 ([~]1400 bp), and 25-42x more reads for rRNA operon (OPR) amplicons ([~]4200 bp). Consequently, sequencing complex communities with OPR primers on ONT is currently not feasible due to the unrealistically high read depth required. This study provides evidence that ONT amplicon sequencing has matured to the point where true ASV-resolved profiling is practically and economically feasible, moving ONT amplicon analysis beyond reliance on OTU clustering or reference alignment to enable application in environments lacking comprehensive reference databases. Key FindingsO_LIIt is now straightforward to generate ASVs on ONT platforms (250-4200 bp) C_LIO_LIONT can resolve intragenomic 16S rRNA gene variants C_LIO_LIASV recovery is successful in both simple and complex communities C_LI