ITS2/18S sequencing for fungi and yeast

ITS2 and 18S rRNA sequencing are key molecular techniques used for identifying and classifying fungi and yeast. Both are DNA-based methods that involve sequencing specific regions within the fungal genome to provide insights into taxonomy, phylogeny, and community structure. These techniques are widely used in environmental microbiology, clinical diagnostics, and biodiversity studies to detect and identify fungi and yeast at the species level.

ITS2 and 18S Regions: An Overview

1. ITS (Internal Transcribed Spacer) Regions:
   • The ITS region consists of two segments: ITS1 and ITS2, which are located between the conserved 18S, 5.8S, and 28S ribosomal RNA (rRNA) genes.
   • The ITS2 region, in particular, is highly variable and often used for fungal and yeast identification because it contains unique sequences that allow for species-level discrimination.
   • The variability in ITS regions allows for the identification of closely related species, making it the gold standard for fungal barcoding.
2. 18S rRNA Gene:
   • The 18S rRNA gene, also known as the small subunit rRNA (SSU rRNA) gene, is a part of the rRNA gene cluster.
   • It is more conserved than ITS regions, making it useful for higher-level taxonomic classifications (e.g., phyla or genus level) but less effective for distinguishing closely related species.
   • 18S rRNA sequencing is often used when researchers are interested in broader phylogenetic relationships within fungi or when studying environmental samples with unknown fungi.

Why Use ITS2 and 18S rRNA for Fungi and Yeast Identification?

• Species Discrimination: The ITS2 region is highly variable and offers sufficient genetic differences to differentiate species within fungal genera. This is especially useful in applications like pathogen identification, biodiversity assessment, and ecological studies.
• Higher-Level Taxonomy: The 18S rRNA gene, being more conserved, is ideal for identifying fungi at higher taxonomic levels, such as orders or families, and is used when broader classification is needed.
• Environmental Studies: Both ITS2 and 18S sequencing are used in metagenomics to study fungal communities in soil, water, and air samples, where a broad range of fungal species and genera are present.

Sequencing Process for ITS2 and 18S rRNA

1. DNA Extraction: Fungal DNA is extracted from pure cultures or environmental samples (e.g., soil, water, or plant tissue). Quality DNA extraction is crucial for obtaining clear, interpretable sequencing results.
2. PCR Amplification:
   • Specific primers target the ITS2 or 18S rRNA region. Common ITS2 primers include ITS3 and ITS4, which amplify the segment between the 5.8S and 28S genes.
   • 18S primers, such as NS1 and NS4, target the conserved regions flanking the 18S gene.
3. Sequencing: Next-generation sequencing (NGS) or Sanger sequencing is employed, depending on the sample size and diversity.
   • NGS (e.g., Illumina or PacBio) is preferred for environmental samples because it allows for high-throughput sequencing and the detection of diverse fungal populations.
   • Sanger sequencing is more commonly used for single-species identification in clinical or diagnostic applications.
4. Bioinformatics Analysis: The sequencing data are analyzed using bioinformatics pipelines. Reads are processed, and sequence quality is checked. For ITS2, sequences are compared to reference databases such as UNITE, which is a curated database for fungal taxonomy. For 18S, databases like SILVA or GenBank are used.

Applications of ITS2 and 18S Sequencing

1. Fungal Biodiversity Studies:
   • ITS2 and 18S sequencing enable researchers to catalog fungal diversity in various ecosystems, such as forests, agricultural soils, and aquatic environments.
   • ITS2 is especially useful for identifying cryptic or rare fungal species that might be overlooked with morphological methods.
2. Pathogen Identification:
   • Clinical laboratories use ITS2 sequencing to identify fungal pathogens causing infections in humans and animals.
   • The method is essential in diagnosing diseases like aspergillosis, candidiasis, and cryptococcosis by accurately identifying pathogenic fungi.
3. Ecological Research and Soil Health:
   • Fungal communities play crucial roles in nutrient cycling, plant growth, and soil health. ITS2 and 18S sequencing help characterize these communities, providing insights into ecosystem dynamics and soil health indicators.
   • These data can guide sustainable agriculture practices by identifying beneficial fungi, such as mycorrhizal fungi, and understanding the impact of agricultural practices on soil biodiversity.
4. Food and Beverage Industry:
   • Yeasts play a significant role in food and beverage production, including fermentation processes in baking, brewing, and winemaking. ITS2 and 18S sequencing are used to monitor yeast diversity and detect potential contaminants.

Advantages and Limitations of ITS2 and 18S Sequencing

Advantages

• High Specificity: ITS2 sequencing provides high resolution for fungal species identification, making it the standard for fungal DNA barcoding.
• Broad Applicability: ITS2 and 18S rRNA sequences are universally present in fungi, making them applicable to a wide range of fungal species.
• High-Throughput Capabilities: NGS enables the rapid sequencing of large numbers of samples, which is useful for studying complex environmental samples or microbiomes.

Limitations

• Database Limitations: Identification accuracy relies on comprehensive and accurate reference databases. Limited or biased databases can lead to misidentification, especially for rare or cryptic fungi.
• Conserved Regions in 18S: The 18S gene’s high conservation limits its utility for species-level identification, meaning it may not be effective for distinguishing closely related species.
• PCR Bias: The choice of primers can introduce biases, as certain primers may not bind equally across all fungal species, leading to underrepresentation of some groups.

ITS2 vs. 18S: Choosing the Right Marker

• Species-Level Identification: ITS2 is preferred when precise species identification is required, such as in clinical diagnostics or detailed biodiversity assessments.
• Broad Phylogenetic Analysis: The 18S region is used when studying phylogenetic relationships among broader taxonomic groups or for general surveys of fungal diversity, especially when exact species identification is less critical.

Future Directions

• Meta-Analysis of Fungal Communities: Advances in bioinformatics and the development of more comprehensive databases will improve the accuracy of ITS2 and 18S sequencing in environmental fungal studies.
• Single-Cell and Multi-Gene Sequencing: Combining ITS2 and 18S sequencing with other molecular markers, such as the LSU (large subunit) rRNA or other protein-coding genes, can provide more robust phylogenetic insights and help resolve ambiguities in species identification.
• Automated Pipelines and Machine Learning: Integrating machine learning into fungal DNA analysis pipelines may enhance identification accuracy, especially when analyzing complex datasets from diverse ecosystems or clinical samples.

ITS2 and 18S rRNA sequencing are powerful tools in fungal and yeast identification, allowing researchers to unravel the diversity, taxonomy, and ecology of these organisms. While each region serves a unique purpose, combining them in a multi-gene approach offers a comprehensive understanding of fungal communities. These sequencing techniques continue to advance, providing essential insights into fungal roles in ecosystems, human health, and various industrial applications.