A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable ‘from benchtop-to-desktop’ metabarcoding workflow to investigate the diet of invertebrate-eaters. We applied our workflow to fecal DNA samples of an invertebrate-eating fish species. A fragment of the COI gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of non-arbitrary thresholds established from control samples and from molecular replicates in order to address the elimination of cross-contamination, PCR/sequencing errors and mistagging artifacts. This resulted in a conservative and informative metabarcoding dataset. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduce...