Abstract
The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.
Highlights
Deep sequencing technologies and DNA barcoding are being increasingly applied to catalog and classify biodiversity
A total of 83,120 full-length internal transcribed spacer (ITS) sequences from kingdom Fungi were obtained from the UNITE database
The sequences belonging to Basidiomycota contained Agaricomycotina (20,522 sequences), Pucciniomycotina (1,951 sequences), Ustilaginomycotina (400 sequences) and unclassified Basidiomycota (808 sequences) (S1 Table). The databases for these sequences are hereafter referred to as insilicoITS, insilicoITS1, and insilicoITS2, with the appropriate prefixes designated by taxonomic grouping (S1 Table)
Summary
Deep sequencing technologies and DNA barcoding are being increasingly applied to catalog and classify biodiversity. Because of the read length limitations of pyrosequencing or Illumina sequencing, only part of the ITS region is usually used, e.g., ITS1 or ITS2. These subregions have been successfully applied in the characterization of fungal communities in some complex ecosystems by the most widely used high-throughput sequencing (including pyrosequencing and Illumina platforms) and have revealed unexpectedly high fungal diversities [1, 11,12,13,14,15]. ITS1 and ITS2 are likely to be the prime targets for the evaluation of fungal diversity through deep sequencing [16]
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