SSU and metagenomics Flashcards
role of SSU rRNA molecules and their impact on revolutionising taxonomy, metagenomics and its principles
taxonomy and microbial classification
What is taxonomy?
The classification of living forms.
taxonomy and microbial classification
Why is Taxonomy Important?
Provides a reference for identifying microbes.
Serves as a universal language for scientists.
Key Taxonomic Terms
Taxonomy: Categorizing organisms.
Taxa: Groups showing similarity.
Phylogeny: Evolutionary history of organisms.
How are Microbes Classified?
Bergey’s Manual of Determinative Bacteriology (1923).
Classification based on physical & biochemical characteristics, not evolutionary relatedness.
Bacterial Identification Criteria
Cell wall composition (Gram-positive vs. Gram-negative).
Morphology (cell shape & colony appearance).
Differential staining.
Oxygen requirements.
Biochemical tests.
Molecular Phylogeny & SSU rRNA
What is Molecular Phylogeny?
Uses DNA sequences to conclude evolutionary relationships.
Molecular Phylogeny & SSU rRNA
Carl Woese’s Discovery (1970s)
rRNA sequences can be used to determine evolutionary relationships.
Molecular Phylogeny & SSU rRNA
What are SSU rRNA Molecules?
Small subunit ribosomal RNA found in all domains of life.
16S rRNA (prokaryotes), 18S rRNA (eukaryotes).
Molecular Phylogeny & SSU rRNA
Structure of Ribosomes
Composed of Large (LSU) & Small Subunit (SSU).
Contains proteins + rRNA
Molecular Phylogeny & SSU rRNA
Why Use SSU rRNA for Phylogeny?
Universally distributed.
Functionally constant.
Highly conserved (slow to change).
Long enough for analysis.
Molecular Phylogeny & SSU rRNA
What is LUCA?
Last Universal Common Ancestor – the origin of all life.
Molecular Phylogeny & SSU rRNA
How Many SSU rRNA Sequences Exist?
Over 2.3 million sequences have been analyzed since 1977.
16S rRNA Gene in Bacterial Phylogeny
Why is 16S rRNA Used in Bacteria?
1500 base pairs long (adequate length).
Has conserved & variable regions.
16S rRNA Gene in Bacterial Phylogeny
Variable Regions in 16S rRNA
Species-specific regions help in identification.
9 variable regions can be used alone or in combination.
16S rRNA Gene in Bacterial Phylogeny
Advantages of 16S rRNA Gene Sequencing
Cheap, suitable for large sample sizes.
Large databases available for comparison.
16S rRNA Gene in Bacterial Phylogeny
Limitations of 16S rRNA Gene Sequencing
Only detects bacteria & archaea (not viruses, fungi, etc.).
PCR bias – some bacteria may be missed.
Low taxonomic resolution (may not identify species/strain).
16S rRNA Gene in Bacterial Phylogeny
Example Use of 16S rRNA Gene Sequencing
Gut microbiota composition studies.
The Great Plate Count Anomaly
What is the Great Plate Count Anomaly?
99% of bacteria from natural environments cannot be cultured in the lab.
The Great Plate Count Anomaly
How to Identify Unculturable Bacteria?
16S rRNA gene sequencing helps identify all bacteria in a sample.
The Great Plate Count Anomaly
Can Genomics Solve the Great Plate Count Anomaly?
Yes, genomic sequencing can identify bacteria without culturing.
16S rRNA Gene Sequencing Method
Step 1: Extract DNA
Collect total DNA from a sample.
16S rRNA Gene Sequencing Method
Step 2: PCR Amplification
Amplify variable regions of the 16S rRNA gene using primers.
16S rRNA Gene Sequencing Method
Step 3: DNA Sequencing
Determine the sequence of amplified DNA.
16S rRNA Gene Sequencing Method
Step 4: Compare to Databases
Identify bacterial species by matching sequences.
PCR in 16S rRNA Gene Sequencing
What is PCR (Polymerase Chain Reaction)?
A technique to amplify specific DNA sequences.
PCR in 16S rRNA Gene Sequencing
Key PCR Components
DNA sample (contains microbial DNA).
Primers (specific for 16S rRNA gene).
Nucleotides (dATP, dCTP, dGTP, dTTP).
DNA polymerase (synthesizes new DNA).
Buffer (for reaction stability).
PCR in 16S rRNA Gene Sequencing
PCR Steps
Denaturation (high temp breaks DNA strands).
Annealing (primers bind to target DNA).
Extension (new DNA strands synthesized).
30-40 cycles per PCR reaction.
Metagenomics
What is Metagenomics?
Sequencing ALL genetic material in a sample (not just 16S rRNA).
Metagenomics
Key Features of Metagenomics
Identifies bacteria, archaea, fungi, viruses.
Analyzes both taxonomic composition & functional potential.
Metagenomics
Technology Used in Metagenomics
Whole Genome Sequencing (WGS) – sequences entire microbial genomes.
Metagenomics
Advantages of Metagenomics
More comprehensive than 16S rRNA sequencing.
Reveals functional genes (e.g., antibiotic resistance).
Metagenomics
Limitations of Metagenomics
Expensive & computationally intensive.
Applications of 16S rRNA Gene Sequencing & Metagenomics
Human Gut Microbiome Studies
16S rRNA: Identified dominant bacterial groups (Bacteroidetes & Firmicutes).
Metagenomics: Revealed functional genes (e.g., metabolism, antibiotic resistance).
Applications of 16S rRNA Gene Sequencing & Metagenomics
Marine Microbial Communities
Metagenomics uncovered previously uncultured microbes & their roles in biogeochemical cycles.
Applications of 16S rRNA Gene Sequencing & Metagenomics
Antibiotic Resistance in Hospitals
Metagenomics identified resistance genes and their potential spread among pathogens.
