Accounting for Intra- and Intergenomic Sequence Variation in Reference Barcodes Improves eDNA Metabarcoding...

Area of Science:

• Marine Biology
• Genomics
• Biodiversity Science

Background:

• Environmental DNA (eDNA) metabarcoding offers rapid biodiversity assessment but is hampered by incomplete reference databases.
• Accuracy is further challenged by intra- and interspecific DNA sequence variation.

Purpose of the Study:

• To evaluate the impact of comprehensive reference databases, including intragenomic and intergenomic variation, on eDNA biodiversity assessments.
• To improve the accuracy and resolution of eDNA metabarcoding for coral reef ecosystems.

Main Methods:

• Collected coral tissue and water samples for reference barcoding and eDNA metabarcoding.
• Utilized genome skimming to generate 28S barcodes from coral specimens.
• Incorporated local reference barcodes and analyzed intragenomic 28S lineages.

Main Results:

• Incorporating local reference barcodes improved ASV (Amplicon Sequence Variant) taxonomic classification, increasing genus-level resolution and correcting misclassifications.
• Excluding intragenomic variation led to unclassified ASVs, highlighting the importance of capturing this variation.
• Integrating reference genome skimming and eDNA metabarcoding significantly expanded known coral family richness, especially at greater depths.
• eDNA metabarcoding detected previously unknown coral families, demonstrating its utility for biodiversity discovery.

Conclusions:

• Incomplete reference databases lead to unclassified ASVs, false positives, and misclassifications in eDNA studies.
• Comprehensive reference databases that account for genomic variation are crucial for accurate eDNA-based biodiversity assessments.
• The integration of genome skimming and eDNA metabarcoding enhances biodiversity discovery, particularly in underexplored deep-sea environments.