An efficient and robust laboratory workflow and tetrapod database for larger scale environmental DNA studies

Area of Science:

• Ecology
• Genetics
• Bioinformatics

Background:

• Environmental DNA (eDNA) metabarcoding is increasingly used for species detection.
• Key challenges include sample contamination and taxonomic misassignment due to DNA degradation and PCR amplification.
• Developing robust methods is crucial for reliable eDNA applications.

Purpose of the Study:

• To present a co-designed lab workflow and bioinformatic pipeline for eDNA metabarcoding.
• To mitigate risks of sample contamination and taxonomic misassignment in eDNA studies.
• To enhance the reliability and applicability of eDNA for ecological and conservation research.

Main Methods:

• A 4-step high-throughput workflow: technical replication, multi-marker use (12S, 16S, CytB), twin-tagging PCR, and PROTAX probabilistic assignment.
• Development of a protocol for curating reference sequence datasets to improve taxonomic accuracy.
• Bioinformatic pipeline for amplicon processing, PROTAX assignment, and twin-tagging validation.

Main Results:

• The workflow successfully minimized contamination and misassignment risks.
• Sequence curation led to the removal of over 50% of public reference sequences for some groups.
• Application to leech DNA data identified 162-190 vertebrate detections from 95-109 samples.

Conclusions:

• The developed metabarcoding workflow enhances result robustness for eDNA research.
• Facilitates wider adoption of eDNA for ecological and conservation insights.
• Highlights the value of eDNA, particularly from invertebrates, for monitoring tetrapod populations.