Abstract
This paper presents an in silico analysis to assess the current state of the fungal UNITE database in terms of the two eukaryote nuclear ribosomal regions, Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2), used in describing fungal diversity. Microbial diversity is often evaluated with amplicon-based high-throughput sequencing approaches, which is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of the primers used for amplification. The goal of this study is to validate if the mismatches of the primers on the binding sites of the targeted taxa could explain the differences observed when using either ITS1 or ITS2 in describing airborne fungal diversity. Hence, the choice of the pairs of primers for each barcode concur with a study comparing the performance of ITS1 and ITS2 in three occupational environments. The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2. However, the database contains an equal number of unidentified taxa from ITS1 and ITS2 regions in the six taxonomic levels employed (phylum, class, order, family, genus, species). The chosen ITS primers showed differences in their ability to amplify fungal sequences from the UNITE database. Eleven taxa consisting of Trichocomaceae, Dothioraceae, Botryosphaeriaceae, Mucorales, Saccharomycetes, Pucciniomycetes, Ophiocordyceps, Microsporidia, Archaeorhizomycetes, Mycenaceae, and Tulasnellaceae showed large variations between the two regions. Note that members of the latter taxa are not all typical fungi found in the air. As no universal method is currently available to cover all the fungal kingdom, continuous work in designing primers, and particularly combining multiple primers targeting the ITS region is the best way to compensate for the biases of each one to get a larger view of the fungal diversity.
Highlights
Bioaerosols are airborne biological particles found almost everywhere, and they are influenced by the surrounding sources [1]
The aim of this study (2) was to evaluate (1) the current state of the latest version of the UNITE database in terms of representative sequences of ITS1 and ITS2, (2) if the mismatches of the primers in the binding sites of the targeted taxa could explain the differences between the two barcodes applying an in silico analysis
The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2
Summary
Bioaerosols are airborne biological particles found almost everywhere, and they are influenced by the surrounding sources [1]. These particles are composed of a mixture of dead and living organisms (e.g., viruses, bacteria, fungi) and their components (e.g., toxins, proteins, metabolites) [2]. Researchers relied on the isolation of fungal species using culture based methods. Only culturable fungal species collected from air samples to study their concentration and diversity induce a bias. The gap in the literature induced by the application of cultivation methods solely is linked to the specific representation of the cultivable and viable portion of fungal aerosols. It is necessary to consider the tip of the iceberg, but look at the bigger picture of fungal aerosols
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