Multiplex PCR targeting lineage specific SNPs ‐ A highly efficient and simple approach to block out predator sequences in molecular gut content analysis

Background: Food webs form the basis of biological communities, though empirical research has been hindered by difficulties in quantifying interactions. Metabarcoding from predator gut content extractions with universal primers promises to provide simple and rapid insights into food web interactions. However, the highly overabundant predator DNA often completely outcompetes that of the digested prey DNA during PCR, impeding the ability to assess the abundance and diversity of prey items.

Methods: Focusing on the issue of overabundance of predator DNA amplified by a commonly used COI primer pair, we use predator lineage-specific SNPs at the 3’-end of PCR primers to selectively block out predators from amplification. While this approach largely prevents predator amplification, it retains high taxonomic versatility for prey lineages. We introduce a novel multilocus assay, targeting four nuclear and mitochondrial rDNA markers and test our approach in a diverse set of spiders from 12 fam...