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Disparities in second‐generation DNA metabarcoding results exposed with accessible and repeatable workflows
Author(s) -
Divoll Timothy J.,
Brown Veronica A.,
Kinne Jeff,
McCracken Gary F.,
O'Keefe Joy M.
Publication year - 2018
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12770
Subject(s) - biology , ion semiconductor sequencing , dna sequencing , taxon , environmental dna , pyrosequencing , illumina dye sequencing , computational biology , evolutionary biology , ecology , genetics , dna , biodiversity , gene
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 OTU s. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTU s were detected by both platforms, 176 (59%) OTU s were detected by the Illumina MiSeq system only, and 17 (6%) OTU s 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.