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The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species
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  • Eva Lena Estensmo,
  • sundy Maurice,
  • Luis Morgado,
  • Pedro Martin-Sanchez,
  • Inger Skrede,
  • Håvard Kauserud
Eva Lena Estensmo
University of Oslo Faculty of Mathematics and Natural Sciences
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sundy Maurice
University of Oslo Faculty of Mathematics and Natural Sciences
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Luis Morgado
Naturalis Biodiversity Center
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Pedro Martin-Sanchez
University of Oslo
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Inger Skrede
University of Oslo Faculty of Mathematics and Natural Sciences
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Håvard Kauserud
University in Oslo
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Abstract

DNA metabarcoding has become a powerful approach for analyzing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers – like the fungal ITS region, may include considerable intraspecific variation, which can lead to over-splitting of species during DNA metabarcoding analyses. Here we assess the effects of intraspecific sequence variation in DNA metabarcoding, by analyzing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS), coupled with error correction with the software DADA2. All focal species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, often occurring in low frequencies, were likely due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS-based fungal community analyses, but an extra clustering step is needed to approach species-level resolution.

Peer review status:ACCEPTED

14 Aug 2020Submitted to Molecular Ecology Resources
18 Sep 2020Submission Checks Completed
18 Sep 2020Assigned to Editor
01 Oct 2020Reviewer(s) Assigned
22 Oct 2020Review(s) Completed, Editorial Evaluation Pending
23 Oct 2020Editorial Decision: Revise Minor
20 Nov 2020Review(s) Completed, Editorial Evaluation Pending
20 Nov 20201st Revision Received
06 Jan 2021Editorial Decision: Accept