Phylogenetic Authentication of Amplicon Sequence Variants in Single-Specimen Metabarcoding of Tropical Insects

Abstract

High-throughput sequencing (HTS) allows large-scale DNA barcoding of individually tagged specimens (‘megabarcoding’), but deep amplicon sequencing produces a mixture of authentic mitochondrial sequences together with nuclear pseudogenes (NUMTs), environmental and cross-sample contaminants, and sequencing artefacts. Standard approaches relying on read clustering or dominant-read selection often fail to classify these types, leading to incorrect taxonomic identifications and species counts. We developed an authentication framework by integrating abundance filtering, phylogenetic placement and taxonomic congruence. The workflow was applied to 18,533 morphospecies of tropical beetles (Coleoptera) from multiple biogeographic regions, which were imaged for family-level identification, prior to individual Illumina barcoding. Sequencing yielded > 36 million reads and 64,544 unique ASVs, which were evaluated against a reference phylogeny of > 13,000 mitogenomes. Authentication succeeded for 86.5% of quality-passing specimens (15,901 ASVs). Non-authentic sequences were technical artefacts (58.0%), environmental contamination including prey DNA (14.2%), intra-individual variants (NUMTs, heteroplasmy; 11.3%) and cross-sample contamination (7.5%). Authentication success and the proportions of failure categories varied markedly across trap types, sampling campaigns, taxonomic groups and sequencing runs. We identified 930 confirmed NUMTs based on consistent co-occurrence patterns and phylogenetic proximity to authenticated haplotypes. Single-specimen HTS data contain substantial biological and technical complexity not resolved by standard filtering methods. Our pipeline-agnostic, phylogenetically informed authentication framework achieves robust recovery of validated barcodes while retaining informative secondary variants, improving the accuracy of molecular ASV data to a standard sufficient for inclusion in barcode reference databases and the phylogenetically informed DNA barcoding of tropical insects.