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 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.

Spatial and Temporal Genetic Diversity and Population Structure of Hemileia vastatrix from Peruvian Coffee Plantations

“…The internal transcribed spacer of the rDNA has been proposed by Schoch et al (2012) as a main barcode marker for fungi to the Consortium for the Barcode of Life, in addition, the use of the rDNA-ITS as an effective marker has been highlighted for differentiating not only at the species level but also among individuals in a population ( Bradshaw et al, 2020 ; Estensmo et al, 2021 ; Nilsson et al, 2008 ; Wu et al, 2018 ). However, its use has been questioned because different levels of intragenomic variation have been reported in a wide range of taxa.…”

Analysing indoor mycobiomes through a large‐scale citizen science study in Norway

“…The demultiplexed R1 and R2 reads were kept separate for the next analyses using dada2 version 1.12 (Callahan et al, 2016): (i) quality filtering and trimming, (ii) dereplication, (iii) generating error models and denoising, (iv) merging in contigs, (v) creating the table of amplicon sequence variants (ASVs) and (vi) removal of chimeras. Additional clustering of ASVs in operational taxonomic units (OTUs), as recommended in previous studies (Estensmo et al, 2021), was done using vsearch version 2.11.1 (Rognes et al, 2016) at 98% similarity. This clustering level is similar to the 98.5% level used to define the species hypotheses (SHs) in the UNITE database (Kõljalg et al, 2013).…”

Using High-Throughput Amplicon Sequencing to Evaluate Intragenomic Variation and Accuracy in Species Identification of Cordyceps Species

“…Amplicon sequencing can extract allelic variants within the genome [ 69 ]. In metabarcoding, high intraspecific (and intragenomic) variation within nrITS is highly problematic as the diversity will be overestimated by treating every haplotype as a biological entity in downstream statistical analysis [ 8 , 44 ]. The problematic in our study is slightly different.…”

“…A Minimum Spanning Network (MSN) method was used from the aligned nrITS data set of individual species using default settings and Ɛ value set at 0. Augmenting the Ɛ value from 0 to 10 increased the pattern complexity for the interconnected node of the different grouping, which led to difficulty in interpreting the haplotype network [ 43 , 44 ]. Combined data (PacBio consensus sequences and Sanger sequences) were used to infer haplotype networks map presented in this study.…”