A scalable and cost-effective nanopore workflow for 16S rRNA microbiome profiling validated in a zebrafish gut model

Advancements in long-read sequencing technologies, such as those developed by Oxford Nanopore Technologies (ONT), have opened new possibilities for 16S rRNA gene profiling of microbial communities by enabling full-length sequencing. In this study, we optimized and validated a cost-effective, in-house ONT workflow for zebrafish gut microbiome analysis and compared its performance to the gold-standard Illumina NovaSeq platform. Using a PCR-based barcoding strategy, we successfully sequenced both the V3-V4 and V1-V9 regions of the 16S rRNA gene, achieving high read accuracy and balanced demultiplexing across samples. Our results demonstrate that ONT short-read (V3-V4) sequencing using the latest V14 chemistry and Dorado basecaller achieves resolution comparable to that of Illumina, supporting amplicon sequence variant classification. Rarefaction analysis confirmed that the sequencing depth (~240,000 reads/sample) was sufficient to capture microbial diversity in the zebrafish gut, a complex and taxonomically rich environment. Furthermore, taxonomic classifications and diversity metrics were consistent between platforms, supporting the reliability of ONT for microbiota profiling. While full-length (V1-V9) ONT sequencing yielded higher taxonomic richness through operational taxonomic units, it did not significantly improve species-level resolution, likely due to current database and bioinformatics limitations. Our custom library preparation protocol simplified multiplexing, reduced costs and improved scalability without compromising accuracy. Although 15 samples were conservatively multiplexed per flow cell in this study, the workflow has since been applied to larger-scale datasets with up to 75 samples per run and to additional amplicon targets, demonstrating its portability and adaptability across diverse biological contexts. These findings position ONT as a viable, scalable and field-deployable alternative to Illumina for microbiome research.