A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts

Abstract

The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric—artificially joined—DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

A survey of xerophilic Aspergillus from indoor environment, including descriptions of two new section Aspergillus species producing eurotium-like sexual states

“…The reference sets include sequences for multiple genes obtained from ex-type cultures for all accepted species in these genera Visagie et al 2014b;Yilmaz et al 2014) and are invaluable as anchoring points for species. Curating databases is laborious and has many complications, but both UNITE and NCBI have ongoing curation projects involving ITS barcodes (Kõljalg et al 2013;Nilsson et al 2015;Schoch et al 2014). ITS will always have limited resolution for species identification.…”

Top 50 most wanted fungi

“…In analogy, for sequence types found only in a single study, some sound skepticism is perhaps in place given the sequence quality-related issues involved in studies based on cloning as well as next-generation sequencing (Hyde et al 2013;Lindahl et al 2013;Hughes et al 2015). However, there are examples to the contrary for both of these situations: sequence types found only in one particular study have proved to be authentic, and "species" found in several different studies have proved to be chimeras (Brown et al 2015;Nilsson et al 2015). This search parameter offers some degree of flexibility by allowing the user to specify whether the number of sequences or the number of studies should be used to order the list of compound clusters.…”

What do we learn from cultures in the omics age? High-throughput sequencing and cultivation of leaf-inhabiting endophytes from beech (Fagus sylvatica L.) revealed complementary community composition but similar correlations with local habitat conditions

“…material 1) and is also detailed in Unterseher et al (2016) and Eusemann et al (2016). Extraction of ITS1 (forward R1 Illumina reads) was done with ITSx (Bengtsson-Palme et al 2013) followed by reference-based chimera checking (Nilsson et al 2015), open-reference OTU picking (complete-linkage clustering at 97% similarity; Rideout et al 2014), selection of representative sequences and taxon assignment (Kõljalg et al 2013). These last steps were also performed with all high-quality Sanger sequences to guarantee best comparability.…”