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Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment
Anastasija Zaiko1,2, Paul Greenfield3,4, Cathryn Abbott5
1Coastal and Freshwater Group, Cawthron Institute, Nelson, New Zealand.
Molecular Ecology Resources
|August 16, 2021
Summary
High-throughput sequencing (HTS) revolutionizes marine biodiversity assessment. Despite lab variations, HTS metabarcoding accurately identifies species and geographic origins, aiding future standardization efforts.
Area of Science:
- Marine biology
- Genomics
- Environmental monitoring
Background:
- High-throughput sequencing (HTS) offers rapid, holistic species detection in marine environments.
- HTS is increasingly used for biodiversity assessments globally.
- Variability in lab procedures and bioinformatics hinders result comparability.
Purpose of the Study:
- To assess the consistency of metabarcoding results using identical samples and primers across different laboratory procedures.
- To identify critical methodological factors causing variability in HTS biodiversity assessments.
Main Methods:
- An international experiment distributed identical biofouling samples to 12 labs.
- Labs used one of two HTS library preparation workflows with standardized DNA extraction, primers, and bioinformatics.
- Technical variables like amplification protocols and instruments were allowed to vary.
Main Results:
- Despite raw data variation, samples consistently grouped by geographical origin.
- Post hoc data cleanup improved classification: 92.81% for 18S rRNA and 95.58% for COI after contamination correction.
- Preservation buffer, sample defrosting, and PCR components were key variability factors.
Conclusions:
- HTS metabarcoding shows robust geographic signal despite methodological variations.
- Standardizing critical factors like preservation and PCR reagents is essential for reliable marine biodiversity monitoring.
- This study provides a foundation for harmonizing HTS protocols in ecological research.
