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

ABSTRACT: 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 co

SUBMITTER: He T 

PROVIDER: S-EPMC12923166 | biostudies-literature | 2026 Feb

REPOSITORIES: biostudies-literature