Disparities in second-generation DNA metabarcoding results exposed with accessible and repeatable workflows

Area of Science:

• Ecology
• Genomics
• Bioinformatics

Background:

• DNA metabarcoding is crucial for identifying prey in predator diets.
• Second-generation sequencing (SGS) technologies may introduce taxon-specific biases.
• Understanding these biases is vital for accurate ecological assessments.

Purpose of the Study:

• To compare prey recovery differences between Ion Torrent PGM and Illumina MiSeq platforms.
• To assess the impact of sequencing workflow on DNA metabarcoding results from bat guano.
• To enhance transparency and repeatability in ecological monitoring methods.

Main Methods:

• Bat guano DNA extracts were used for metabarcoding.
• The arthropod cytochrome c oxidase I (COI) minibarcode region was targeted.
• Sequencing was performed on both Ion Torrent PGM and Illumina MiSeq platforms.
• A standardized analysis pipeline with a high OTU clustering threshold (≥98.5%) and copy number filtering was applied.

Main Results:

• Illumina MiSeq detected significantly more prey Operational Taxonomic Units (OTUs) and insect orders compared to Ion Torrent.
• Of 297 unique prey taxa identified, 59% were exclusively detected by Illumina MiSeq, while only 6% were exclusive to Ion Torrent.
• Both platforms yielded similar costs, but Illumina MiSeq generated six times more reads.

Conclusions:

• Sequencing platform choice influences prey detection in DNA metabarcoding studies.
• Illumina MiSeq demonstrated superior performance in prey recovery from bat guano.
• Standardized approaches and platform considerations are essential for reliable long-term ecological monitoring.