Discovering Patterns in the Microbiome

Overview

• Focus on 16s variable regions and their role in studying microbiomes.
• Ribosomal RNA (rRNA) is commonly used to characterize microbiomes.
• Introduction to the central dogma of molecular biology: DNA → RNA → Protein.

Central Dogma of Molecular Biology

• Coined by Francis Crick: Molecular biology involves the transfer of sequential information (DNA to RNA to Protein).
• DNA makes RNA; RNA makes Protein.
• RNA can also function independently, e.g., in ribosomes.

Bacterial Community and DNA Sequencing

• Shotgun Sequencing: Grinds up DNA into short sequences, leading to diverse gene sequences.
• Marker Gene Sequencing (16s Sequencing): Targets a specific gene common in all organisms (ribosome).
  • Focuses on the 16s gene, a part of the ribosome, approximately 1500 nucleotides long.
  • Used for comparing similar genes across different organisms.

Ribosome and 16s rRNA

• Ribosome: Made of RNA strands and proteins; synthesizes proteins from RNA.
• 16s rRNA is a marker gene for studying microbiomes.
• Structural importance and variability:
  • Conserved regions: Essential for function, mutations can be lethal.
  • Variable regions: Mutations occur without affecting function.
  • Segments are termed V1 to V9, representing variable and conserved regions.

Selecting and Sequencing 16s rRNA

• Select a sequence combining conserved and variable regions for species identification.
• Use DNA probes to isolate and amplify specific 16s rRNA segments for sequencing.
• Commonly sequenced segment is in the variable four region (V4).

Data Analysis

• PCR Process: Amplifies specific DNA segments using probes.
• Clustering Sequences: Group similar sequences (97% similarity) into Operational Taxonomic Units (OTUs).
• Largest sequencing runs can produce millions of sequences reduced to thousands of OTUs.

Limitations and Alternatives

• Marker Gene Sequencing: Limited by the specificity of the gene region sequenced, analogous to having a single puzzle piece.
• Shotgun Sequencing: Provides a comprehensive view, identifying species and genes, but more costly.
• Data interpretation includes species identification, gene presence, and functional pathways.

Conclusion

• 16s sequencing offers a method for detailed microbiome analysis but has limitations compared to shotgun sequencing.
• Understanding different sequencing approaches helps in analyzing and interpreting microbiome data effectively.