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 several genetic markers provides a more comprehensive understanding of microbial communities, but it comes with challenges, such as differences in how abundant certain species are and difficulties in sequencing.
Eukaryotic Sequencing Challenges: There are several issues to address:
Improving the efficiency of DNA recovery.
Creating accurate databases for identifying specific organisms.
Differentiating between similar forms of organisms (teleomorph vs anamorph).
Choosing the appropriate genetic marker for study.
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.