Sorting specimen-rich invertebrate samples with cost-effective NGS barcodes: Validating a reverse workflow for...

Area of Science:

• Molecular Biology
• Taxonomy
• Bioinformatics

Background:

• Traditional specimen sorting by morphology is laborious and prone to error.
• Molecular methods like metabarcoding can destroy valuable voucher specimens.
• Existing DNA barcoding workflows are often too expensive for high-throughput applications.

Purpose of the Study:

• To develop and validate a cost-effective next-generation sequencing (NGS) barcoding pipeline for high-throughput specimen analysis.
• To assess the feasibility of a 'reverse workflow' for presorting specimens using DNA barcodes.
• To accelerate invertebrate species discovery and facilitate taxonomic research.

Main Methods:

• Developed an NGS barcoding pipeline for generating short (313 bp COI) specimen-specific DNA barcodes at low cost (<$0.50 per specimen).
• Applied the pipeline to a large sample of tropical ants, successfully obtaining barcodes for 82% of specimens.
• Sorted specimens into molecular operational taxonomic units (mOTUs) using objective clustering and Automated Barcode Gap Discovery (ABGD).

Main Results:

• Generated high-throughput, cost-effective DNA barcodes for thousands of individual specimens.
• Identified 88-90 molecular operational taxonomic units (mOTUs) with high agreement to morphospecies (0.95 match ratio at 4% clustering).
• Discovered 48 new species barcodes, with 28 potentially new to science, highlighting the utility for species discovery.

Conclusions:

• The NGS barcoding pipeline offers a technically simple and affordable solution for large-scale specimen barcoding.
• This approach significantly accelerates invertebrate species discovery and aids in building comprehensive DNA barcode databases.
• The method provides precise abundance information and facilitates downstream taxonomic validation and research.