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

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Illumina MiSeq recovered more prey taxa and insect orders than Ion Torrent in bat guano DNA metabarcoding. This highlights sequencing biases impacting ecological monitoring and long-term community change detection.

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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.