Effective metabarcoding of endophytic fungi through host-exclusive primer design for nanopore long-read sequencing

Metabarcoding is a powerful tool to simultaneously identify multiple taxa within a DNA sample. However, despite its power, metabarcoding poses challenges when applied to host-associated microbiomes, primarily due to the substantial co-amplification of host DNA. Here we developed a host-exclusive primer design workflow, to selectively generate amplicons from target taxa while excluding the host. This workflow is centered around a new computational tool, mbc-prime, that can generate a list of discriminative candidate primers and score them. We showcase the use of this tool in the process of designing primers for the long-read metabarcoding of endophytic fungi in Vinca minor. Four new forward and two new reverse primers reliably amplify large regions of the fungal rDNA of a fungal mock community and the extracted holobiont genomic DNA. Primers exhibit high specificity for fungal rDNA and robust amplification across a diverse range of fungal taxa: with the best primer pairs, we produced fungal amplicons free of plant DNA. Our workflow can be used to study the composition of complex host-associated microbiomes. This workflow should furthermore be universally applicable for the design of discriminative primers in a user-friendly and practical manner and thus be of use for various researchers in microbiome research. https://doi.org/10.1101/2024.11.25.625223