Lecture 22:

Exploring the ITS Gene Amplicon Sequencing for Microbial Community Studies

Introduction to ITS Gene Amplicon Sequencing

Microbial Community Members

• Bacteriophage, Archaea, Protozoa, Fungi, and Bacteria: These form the major classifications within microbial communities, with varying biomass contributions.

The Kingdom of Fungi

Overview

• Species Estimate: Estimated 2.5 – 3.5 million species, with around 148,000 described.

• Pathogenic Profiles: Includes approximately 8,000 plant pathogens and 300 human or animal pathogens.

Characteristics of Fungi and Yeasts

• Eukaryotic and Spore-bearing: Fungi are heterotrophic and produce extracellular enzymes to absorb nutrients.

• Diverse Functions: Includes roles in food production, spoilage, antibiotic production, bioprocessing, and potential pathogenicity to humans and animals.

Fungi in the Human Microbiome

Human Body Mycobiota

• Fungal Colonizers: Fungi like Candida, Saccharomyces, and Cladosporium are commonly found in the human gut, influenced by diet, immune response, and microbial interactions.

• Factors influencing the mycobiome include: Skin polymers inaccessible to fungi, don’t tolerate 37C, prefer low pH, oxygen gradients, bacterial-fungal relationships.

Mycobiome Research

• Research Gaps: While bacterial microbiome research is extensive, mycobiome research is less developed due to factors such as species abundance, sequencing challenges, and available databases.

The Kingdom of Protists

Characteristics of Protists

• Eukaryotic and Unicellular: Protists can be heterotrophic or photosynthetic and play crucial roles in nutrient cycles; producers (algae) or consumers (protozoa). In the rumen, ciliates contribute significantly to feed efficiency and methane production.

Culture-Based Approach Limitations

• Challenges: Culture techniques for ciliated protozoa from the rumen face significant challenges, making advanced sequencing approaches vital for accurate analysis.

Sequencing and Targeting Marker Genes

Targeting the 18S rRNA Gene

• Advantages: Allows for broad classification of eukaryotic organisms, particularly protozoa and fungi. High-throughput, allowing for more phylogenic classification rather than intraspecies genetic diversity.

• Disadvantages: Lower resolution at the species level; can’t distinguish closely related species well; varies in sensitivity. Exhibits less diversity, copy number varies, annotation limited to current reference libraries.

Internal Transcribed Spacer (ITS) Sequencing

• Target Regions: ITS regions, including ITS1, ITS2, and the 5.8S ribosomal RNA gene, provide more detailed genetic information helpful in identifying fungal species.

Comparative Analysis of Marker Genes

• Comparison Studies: Studies comparing ITS regions with 18S and other genomic targets reveal the relative accuracy and potential biases in fungal community identification.

Leveraging Amplicon Sequencing Across Kingdoms

• Holistic Insight: Using multiple markers allows for a broader view but introduces caveats like varying abundances and sequencing difficulties.

• Challenges for Eukaryotic sequencing: optimizing DNA recovery, assembling databases for taxonomic identification, teleomorph vs anamorph taxonomic confusion, selecting correct marker.

Practical Applications and Differential Abundances

• Microbial Community Development: Studies on microbial communities in rumen inoculation show differential relative abundances of microbial kingdoms over time.

Conclusion

Key Insights from ITS Sequencing

• Fungi vs. Bacteria Stability: Fungal communities exhibit less stability and are more difficult to sample and sequence compared to bacterial communities.

• Diet Influence: Diet profoundly impacts the composition and interaction of gut fungi and bacteria, emphasizing the dynamic nature of microbial ecosystems.

By understanding the intricacies of ITS gene amplicon sequencing and the broader implications of microbial community studies, we can harness these insights for applications in health, agriculture, and environmental sciences.