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

Timothy J. Divoll, Veronica A. Brown, Jeff Kinne, Gary F. McCracken, Joy M. O'Keefe

Research output: Contribution to journalArticlepeer-review


Different second-generation sequencing technologies may have taxon-specific biases when DNA metabarcoding prey in predator faeces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same faecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort towards transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat faecal samples, targeted the arthropod cytochrome c oxidase I minibarcode region and sequenced the product on both second-generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long-term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.

Original languageEnglish (US)
Pages (from-to)590-601
Number of pages12
JournalMolecular ecology resources
Issue number3
StatePublished - May 2018
Externally publishedYes


  • Chiroptera
  • eDNA metabarcoding
  • Illumina MiSeq
  • Ion Torrent PGM
  • R Notebook

ASJC Scopus subject areas

  • Biotechnology
  • Ecology, Evolution, Behavior and Systematics
  • Genetics


Dive into the research topics of 'Disparities in second-generation DNA metabarcoding results exposed with accessible and repeatable workflows'. Together they form a unique fingerprint.

Cite this