Primer-Less Species Identification Throughout Fungal (Tuber magnatum), Plant (Corylus avellana) and Animal (Eisenia fetida) Kingdoms by Direct RNA Sequencing.

Accurate species identification is essential for biodiversity studies, ecological monitoring, and biosecurity, but current molecular approaches rely on PCR amplification, which requires universal primers. Here, we evaluated the possibility of primer-free species identification via direct RNA sequencing (dRNA-seq) by using Oxford Nanopore Technology (ONT). Total RNA isolated from three major eukaryotic kingdoms-fungal (Tuber magnatum Picco 1788), plant (Corylus avellana L. 1753), and animal (Eisenia fetida Savigny 1826) specimens-was sequenced without reverse transcription and PCR amplification. Taxonomic assignments based on similarity to ribosomal nuclear (fungi and animals) and chloroplast (plant) transcripts enabled reliable host species identification. Ribosomal RNA reads dominated the datasets and supported accurate identification of the target organisms, while additional sequences revealed associated microbiota and co-occurring taxa. Notably, dRNA-seq successfully detected symbiotic bacteria in E. fetida and latent fungal infection in C. avellana, highlighting the method sensitivity. However, the complex organization of nuclear ribosomal gene clusters may complicate taxonomic assignment in plants, underscoring the need for improved analytical pipelines. Overall, our results provide proof of concept that dRNA-seq enables primer-independent species identification while simultaneously providing insights into the host microbiomes. Direct sequencing of naturally present RNA molecules generated sufficient sequence information for species identification across different taxonomic kingdoms, indicating the technical feasibility of the developed technique. Further validation using larger biological datasets and direct comparisons with conventional sequencing methods will determine its wider applicability as an innovative and complementary species identification approach.