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

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.

To denoise or to cluster, that is not the question: optimizing pipelines for COI metabarcoding and metaphylogeography

“…Using information at the strain level may be useful in the case of prokaryotes, and in low-variability eukaryote markers such as ribosomal 18S rDNA there may be correspondence between species and unique sequences (indeed, in many cases different species share sequences). But even in more variable nuclear markers such as ITS, a clustering step is necessary [ 58 ]. In eukaryotes the unit of diversity analyses is the species.…”

Artificial selection of stable rhizosphere microbiota leads to heritable plant phenotype changes

“…Total bacterial and fungal diversity and composition were obtained by sequencing the V3‐V4 hypervariable regions of the bacterial 16S rRNA gene (small subunit of the prokaryotic ribosomal operon, Baker et al, 2003; Herlemann et al, 2011) and ITS2 region (Internal Transcribed Spacer 2 in the ribosomal operon of fungi, Ihrmark et al, 2012; White et al, 1990) via Illumina Miseq 2 × 250 bp paired‐end analysis. For consistency, we analysed both the 16S rRNA and ITS2 sequences using an OTU pipeline instead of Amplicon Sequence Variants (ASV) since the latter is currently not optimal for fungal ITS (Estensmo et al, 2021). Raw sequences have been deposited to the SRA public repository (Sequence Read Archive, Bacteria: SUB7720753, Fungi: SUB7738355).…”

DNA barcoding of fungal specimens using long-read high-throughput sequencing

“…Our results suggest that the exact sequence variant (ESV) based approaches (Callahan et al, 2017) are not optimal for species-level metabarcoding analyses of fungal diversity (see also Estensmo et al, 2021; Tedersoo et al, 2022) and perhaps eukaryotes in general (Antich et al, 2021; Porter & Hajibabaei, 2021), by potentially retrieving artefactual taxa. In conclusion, multiplex DNA barcoding of the fungal ITS marker using a PacBio third-generation HTS protocol is a useful tool for taxonomic assessment of large sets of vouchered fungal specimens.…”