The influence of intraspecific sequence variation during DNA metabarcoding: A case study of eleven fungal species

DNA metabarcoding has become a powerful approach for analysing 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, o...

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Bibliographic Details
Published in:Molecular ecology resources 2021-05, Vol.21 (4), p.1141-1148
Main Authors: Estensmo, Eva Lena F., Maurice, Sundy, Morgado, Luis, Martin‐Sanchez, Pedro M., Skrede, Inger, Kauserud, Håvard
Format: Article
Language:English
Subjects:
Clustering
community ecology
Deoxyribonucleic acid
DNA
DNA metabarcoding
Error correction
Fungi
Genetic diversity
Haplotypes
ITS
Local population
Markers
Next-generation sequencing
Nucleotide sequence
Species
Variation
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Summary:DNA metabarcoding has become a powerful approach for analysing 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 oversplitting of species during DNA metabarcoding analyses. Here we assessed the effects of intraspecific sequence variation in DNA metabarcoding by analysing 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 the eleven 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, typically occurring in low frequencies, were probably 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.
ISSN:1755-098X
1755-0998
DOI:10.1111/1755-0998.13329