Fungal Identification by Sequencing: LSU, SSU, and ITS Regions

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The mycobiome plays a vital role in processes such as decomposition, symbiosis, and disease progression. Knowing the role of different fungi in these processes is critical to understanding microbial communities and their impacts on the environment. In drug discovery, accurate fungal identification unlocks information about species and biochemical properties.

Rapid fungal identification entails early diagnosis, treatment, and therapy. For non-specialized laboratories, identification can be complicated, labor-intensive, and time-consuming. Morphological screening is often misleading and challenging. Biochemical tests can be counter-productive while rapid kits are often unreliable and laborious. Delays of appropriate treatment often lead to poor outcomes. Therefore, quick and accurate identification methods are essential features of a diagnostic laboratory. Together with traditional methods, molecular techniques are utilized to identify the phylogeny of important fungi, shown in figure 1. The ribosomal DNA sequence (rDNA), either from the large subunit (LSU) or small subunit (SSU), information can be used for organizing fungi into taxa. The intervening internal transcribed spacer (ITS) regions contain greater sequence variation as they evolve faster which makes them more suitable for strain and species identification than rDNA sequences.

The SSU is used if interest is on the higher taxonomic level identification, while LSU and ITS can be combined to identify genus and species level of identification. However, for species-level identification, the ITS is the most useful because of its ease in amplification, large barcode gap, and it can be sequenced from previously described fungi with no sequence data available by only sequencing the type material. Due to its practicality and wide applicability, The ITS was therefore chosen as the official fungal barcode by the Consortium for the Barcode of Life.

Despite its proven usability and reputation, the ITS fungal barcoding marker has also its limitations. Apparently, it does not work well in some genera, such as Aspergillus, Penicillium, Fusarium, Trichoderma, and Cladosporium which all have narrow or no barcode gaps in their ITS regions. To augment this limitation, protein-coding gene regions could be incorporated into the barcoding analysis as they contain intron regions that can evolve even faster than the ITS region. Additionally, ITS variation occurs intragenomically in around ∼3−5% of fungi belonging to Ascomycota and Basidiomycota. Also, no single lineage-specific value cut-off can be applied across all groups as it is still being determined. There is far more data available for nuclear ribosomal genes than the ITS region; hence, it is encouraged to conduct analysis or narrow down first using rDNA sequences and then switch to the ITS region in case of inconclusive results.

The use of the ITS region for fungal identification has its benefits but it should be used with caution. It is important to note that identification on GenBank BLAST alone is often unreliable due to the presence of unoptimized low-quality sequences. Therefore, curated sequence data from databases such as BOLD, CBS-KNAW, RTL, and ISHT should be consulted for comparison and test of reliability. Through NGS, fungal ITS sequencing has been efficient and easier as culturing constraints are removed.


1. Raja HA, Miller AN, Pearce CJ, Oberlies NH. Fungal identification using molecular tools: a primer for the natural products research community. Journal of natural products. 2017 Mar 24;80(3).

2. Pryce TM, Palladino S, Kay ID, Coombs GW. Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system. Medical mycology. 2003 Oct 1;41(5).


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