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Current rDNA reference sequence databases are tailored towards shorter DNA markers, such as parts of the 16/18S marker or the internally transcribed spacer (ITS) region. However, due to advances in long-read DNA sequencing technologies, longer stretches of the rDNA operon are increasingly used in environmental sequencing studies to increase the phylogenetic resolution. There is, therefore, a growing need for longer rDNA reference sequences. Here, we present the ribosomal operon database (ROD), which includes eukaryotic full-length rDNA operons fished from publicly available genome assemblies. Full-length operons were detected in 34.1% of the 34,701 examined eukaryotic genome assemblies from NCBI. In most cases (53.1%), more than one operon variant was detected, which can be due to intragenomic operon copy variability, allelic variation in non-haploid genomes, or technical errors from the sequencing and assembly process. The highest copy number found was 5947 in Zea mays. In total, 453,697 unique operons were detected, with 69,480 operon variant clusters remaining after intragenomic clustering at 99% sequence identity. The operon length varied extensively across eukaryotes, ranging from 4136 to 16,463 bp, which will lead to considerable polymerase chain reaction (PCR) bias during amplification of the entire operon. Clustering the full-length operons revealed that the different parts (i.e., 18S, 28S, and the hypervariable regions V4 and V9 of 18S) provide divergent taxonomic resolution, with 18S, the V4 and V9 regions being the most conserved. The ROD will be updated regularly to provide an increasing number of full-length rDNA operons to the scientific community., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

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., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Analysis of microbial community structure by multivariate ordination methods, using data obtained by high-throughput sequencing of amplified markers (i.e., DNA metabarcoding), often requires clustering of DNA sequences into operational taxonomic units (OTUs). Parameters for the clustering procedure tend not to be justified but are set by tradition rather than being based on explicit knowledge. In this study, we explore the extent to which ordination results are affected by variation in parameter settings for the clustering procedure. Amplicon sequence data from nine microbial community studies, representing different sampling designs, spatial scales and ecosystems, were subjected to clustering into OTUs at seven different similarity thresholds (clustering thresholds) ranging from 87% to 99% sequence similarity. The 63 data sets thus obtained were subjected to parallel DCA and GNMDS ordinations. The resulting community structures were highly similar across all clustering thresholds. We explain this pattern by the existence of strong ecological structuring gradients and phylogenetically diverse sets of abundant OTUs that are highly stable across clustering thresholds. Removing low-abundance, rare OTUs had negligible effects on community patterns. Our results indicate that microbial data sets with a clear gradient structure are highly robust to choice of sequence clustering threshold., (© 2018 John Wiley & Sons Ltd.)

The formation of chimeric sequences can create significant methodological bias in PCR-based DNA metabarcoding analyses. During mixed-template amplification of barcoding regions, chimera formation is frequent and well documented. However, profiling of fungal communities typically uses the more variable rDNA region ITS. Due to a larger research community, tools for chimera detection have been developed mainly for the 16S/18S markers. However, these tools are widely applied to the ITS region without verification of their performance. We examined the rate of chimera formation during amplification and 454 sequencing of the ITS2 region from fungal mock communities of different complexities. We evaluated the chimera detecting ability of two common chimera-checking algorithms: perseus and uchime. Large proportions of the chimeras reported were false positives. No false negatives were found in the data set. Verified chimeras accounted for only 0.2% of the total ITS2 reads, which is considerably less than what is typically reported in 16S and 18S metabarcoding analyses. Verified chimeric 'parent sequences' had significantly higher per cent identity to one another than to random members of the mock communities. Community complexity increased the rate of chimera formation. GC content was higher around the verified chimeric break points, potentially facilitating chimera formation through base pair mismatching in the neighbouring regions of high similarity in the chimeric region. We conclude that the hypervariable nature of the ITS region seems to buffer the rate of chimera formation in comparison with other, less variable barcoding regions, due to shorter regions of high sequence similarity., (© 2016 John Wiley & Sons Ltd.)

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut-off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut-off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under- or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi., (© 2013 Blackwell Publishing Ltd.)