A discriminative primer design workflow enables selective metabarcoding, demonstrated using long-read sequencing of endophytic fungi

Metabarcoding is a powerful tool to simultaneously identify multiple taxa within a habitat. However, its application to host-associated microbiomes is challenged by 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 design of primers for long-read metabarcoding of endophytic fungi in Vinca minor. Mbc-prime streamlines the design of fungus-specific primers, enabling efficient and plant-free amplification of fungal rDNA from mixed DNA samples. Our workflow can be used to study the composition of complex host-associated microbiomes. It should 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.