AI Insight
Researchers developed OrCa-seq (Oxford Nanopore rRNA and COI amplicon sequencing), a portable and low-cost protocol that simultaneously sequences four genetic amplicons from meiofauna specimens, covering nearly the complete ribosomal RNA cistron and the standard COI barcoding region. The method was validated using freshwater and limno-terrestrial meiofauna isolated by students, processed in 96-well plate format with results available within one day of sample lysis. Results confirmed broad taxonomic applicability across meiofauna groups, though recovery efficiency varied by taxon and amplicon, with the full-length COI Folmer fragment proving the most difficult to recover consistently.
Why it matters
This protocol addresses a longstanding gap in meiofauna biodiversity research by combining the universal coverage of rRNA markers with the species-level resolution of mitochondrial COI in a single, field-deployable workflow. Its adaptability to diverse specimen types and primer combinations positions it as a practical tool for large-scale biodiversity inventories, ecological monitoring, and taxonomic identification programs.
⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
Molecular data have revolutionised taxonomic and ecological research on the hyperdiverse communities of aquatic benthic microinvertebrates known as meiofauna. However, reference sequence databases remain highly incomplete, with variable barcode genes or fragments studied from taxon to taxon. Furthermore, there is a typical tradeoff between universality of primers and phylogenetic resolution, with rRNA markers being robustly recoverable but failing to resolve species-level divergences, and mitochondrial markers showing the reverse trend. Here, we introduce Oxford Nanopore rRNA and COI amplicon sequencing (OrCa-seq), a rapid, low-cost protocol for parallel long-range PCR amplification and multiplexed sequencing of four amplicons, spanning the nearly-complete rRNA cistron (~7-8 kb) and the widely studied Folmer region of COI (represented as overlapping 313 and 658 bp amplicons). This protocol, with its associated bioinformatic workflow, was designed for conducting biodiversity inventories of meiofauna and can be easily carried out in field research and educational contexts, with data available from 96-well plates of specimens within a day of lysis. To validate the method, we processed six plates of student-isolated freshwater and limno-terrestrial meiofauna, characterising the recovery of target genes and taxa with both automated and human-curated BLAST database comparisons. These data demonstrate the universal applicability of OrCa-seq across effectively all meiofauna, including the very smallest species. Nonetheless, recovery efficiency for each amplicon shows variation by taxon, with the full-length Folmer COI amplicon standing out as the most challenging. We present exemplar phylogenetic trees integrating reference sequences, demonstrating the utility of these data in confirming morphological determinations and in identifying anonymous specimens in a reverse taxonomy context. While developed in a specific educational context for use on meiofauna, the OrCa-seq approach should be readily scalable to larger research datasets, adaptable to many specimen types, and to any combination of taxon- or target-specific primers. As such, it represents a compelling multi-locus extension to the ever-growing repertoire of nanopore DNA barcoding protocols.
Source: Portable, multilocus DNA barcoding across the diversity of meiofauna