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

Area of Science:

• Genomics
• Bioinformatics
• Ecology

Background:

• High-throughput sequencing (HTS) enables large-scale DNA barcoding (megabarcoding) but is prone to errors from nuclear pseudogenes (NUMTs), contaminants, and artefacts.
• Standard methods struggle to differentiate authentic sequences from these errors, leading to inaccurate species identification and counts.

Purpose of the Study:

• To develop and validate a robust authentication framework for HTS DNA barcoding data.
• To improve the accuracy of taxonomic identification and species counts in large-scale biodiversity assessments.

Main Methods:

• Integrated abundance filtering, phylogenetic placement, and taxonomic congruence to authenticate DNA barcodes.
• Applied the framework to 18,533 morphospecies of tropical beetles (Coleoptera) using Illumina barcoding.
• Evaluated 64,544 unique amplicon sequence variants (ASVs) against a reference phylogeny of over 13,000 mitogenomes.

Main Results:

• Successfully authenticated 86.5% of quality-passing specimens (15,901 ASVs).
• Identified non-authentic sequences as technical artefacts (58.0%), environmental contamination (14.2%), NUMTs/heteroplasmy (11.3%), and cross-sample contamination (7.5%).
• Confirmed 930 NUMTs and demonstrated significant variation in authentication success across sampling parameters.

Conclusions:

• Standard filtering methods are insufficient for complex HTS data.
• The developed phylogenetically informed framework robustly recovers validated barcodes and improves the accuracy of molecular ASV data.
• This framework enhances the reliability of DNA barcoding data for reference databases and biodiversity studies, particularly for tropical insects.