Full-length 16S rRNA nanopore sequencing enables species resolution of <i>Fusobacterium</i> associated with colorectal cancer

Recent studies have revealed that the long-recognized link between the historically defined <i>Fusobacterium nucleatum</i> group and colorectal cancer is largely driven by <i>Fusobacterium animalis</i>. This species, along with two others (<i>Fusobacterium polymorphum</i> and <i>Fusobacterium vincentii</i>), was recently reclassified as distinct from <i>F. nucleatum</i>, highlighting functional divergence within this group. Due to their close genetic relatedness, traditional partial 16S rRNA gene sequencing lacks the resolution to reliably distinguish these species. Nevertheless, accurate species-level identification remains essential in cancer-associated microbiome research. Here, we demonstrate that full-length 16S rRNA sequencing using Oxford Nanopore Technology, combined with a novel custom demultiplexing software, enables robust species-level discrimination within the <i>Fusobacterium</i> genus. Our approach accurately classified clinically relevant <i>Fusobacterium</i> species and recovered their expected proportions from whole cells, DNA mixtures, and clinical CRC specimens. This method provides high-resolution profiling to elucidate species-specific roles of <i>Fusobacterium</i> in colorectal cancer.