SSU and metagenomics

Question 1

taxonomy and microbial classification

What is taxonomy?

Answer 1

The classification of living forms.

Question 2

taxonomy and microbial classification

Why is Taxonomy Important?

Answer 2

Provides a reference for identifying microbes.
Serves as a universal language for scientists.

Question 3

Key Taxonomic Terms

Answer 3

Taxonomy: Categorizing organisms.
Taxa: Groups showing similarity.
Phylogeny: Evolutionary history of organisms.

Question 4

How are Microbes Classified?

Answer 4

Bergey’s Manual of Determinative Bacteriology (1923).
Classification based on physical & biochemical characteristics, not evolutionary relatedness.

Question 4

Bacterial Identification Criteria

Answer 4

Cell wall composition (Gram-positive vs. Gram-negative).
Morphology (cell shape & colony appearance).
Differential staining.
Oxygen requirements.
Biochemical tests.

Question 4

Molecular Phylogeny & SSU rRNA

What is Molecular Phylogeny?

Answer 4

Uses DNA sequences to conclude evolutionary relationships.

Question 4

Molecular Phylogeny & SSU rRNA

Carl Woese’s Discovery (1970s)

Answer 4

rRNA sequences can be used to determine evolutionary relationships.

Question 5

Molecular Phylogeny & SSU rRNA

What are SSU rRNA Molecules?

Answer 5

Small subunit ribosomal RNA found in all domains of life.
16S rRNA (prokaryotes), 18S rRNA (eukaryotes).

Question 6

Molecular Phylogeny & SSU rRNA

Structure of Ribosomes

Answer 6

Composed of Large (LSU) & Small Subunit (SSU).
Contains proteins + rRNA

Question 7

Molecular Phylogeny & SSU rRNA

Why Use SSU rRNA for Phylogeny?

Answer 7

Universally distributed.
Functionally constant.
Highly conserved (slow to change).
Long enough for analysis.

Question 8

Molecular Phylogeny & SSU rRNA

What is LUCA?

Answer 8

Last Universal Common Ancestor – the origin of all life.

Question 9

Molecular Phylogeny & SSU rRNA

How Many SSU rRNA Sequences Exist?

Answer 9

Over 2.3 million sequences have been analyzed since 1977.

Question 10

16S rRNA Gene in Bacterial Phylogeny

Why is 16S rRNA Used in Bacteria?

Answer 10

1500 base pairs long (adequate length).
Has conserved & variable regions.

Question 11

16S rRNA Gene in Bacterial Phylogeny

Variable Regions in 16S rRNA

Answer 11

Species-specific regions help in identification.
9 variable regions can be used alone or in combination.

Question 12

16S rRNA Gene in Bacterial Phylogeny

Advantages of 16S rRNA Gene Sequencing

Answer 12

Cheap, suitable for large sample sizes.
Large databases available for comparison.

Question 13

16S rRNA Gene in Bacterial Phylogeny

Limitations of 16S rRNA Gene Sequencing

Answer 13

Only detects bacteria & archaea (not viruses, fungi, etc.).
PCR bias – some bacteria may be missed.
Low taxonomic resolution (may not identify species/strain).

Question 14

16S rRNA Gene in Bacterial Phylogeny

Example Use of 16S rRNA Gene Sequencing

Answer 14

Gut microbiota composition studies.

Question 15

The Great Plate Count Anomaly

What is the Great Plate Count Anomaly?

Answer 15

99% of bacteria from natural environments cannot be cultured in the lab.

Question 16

The Great Plate Count Anomaly

How to Identify Unculturable Bacteria?

Answer 16

16S rRNA gene sequencing helps identify all bacteria in a sample.

Question 17

The Great Plate Count Anomaly

Can Genomics Solve the Great Plate Count Anomaly?

Answer 17

Yes, genomic sequencing can identify bacteria without culturing.

Question 18

16S rRNA Gene Sequencing Method

Step 1: Extract DNA

Answer 18

Collect total DNA from a sample.

Question 19

16S rRNA Gene Sequencing Method

Step 2: PCR Amplification

Answer 19

Amplify variable regions of the 16S rRNA gene using primers.

Question 20

16S rRNA Gene Sequencing Method

Step 3: DNA Sequencing

Answer 20

Determine the sequence of amplified DNA.

Question 21

16S rRNA Gene Sequencing Method

Step 4: Compare to Databases

Answer 21

Identify bacterial species by matching sequences.

Question 22

# PCR in 16S rRNA Gene Sequencing What is PCR (Polymerase Chain Reaction)?

Answer 22

A technique to amplify specific DNA sequences.

Question 23

# PCR in 16S rRNA Gene Sequencing Key PCR Components

Answer 23

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).

Question 24

# PCR in 16S rRNA Gene Sequencing PCR Steps

Answer 24

Denaturation (high temp breaks DNA strands). Annealing (primers bind to target DNA). Extension (new DNA strands synthesized). 30-40 cycles per PCR reaction.

Question 25

# Metagenomics What is Metagenomics?

Answer 25

Sequencing ALL genetic material in a sample (not just 16S rRNA).

Question 26

# Metagenomics Key Features of Metagenomics

Answer 26

Identifies bacteria, archaea, fungi, viruses. Analyzes both taxonomic composition & functional potential.

Question 27

# Metagenomics Technology Used in Metagenomics

Answer 27

Whole Genome Sequencing (WGS) – sequences entire microbial genomes.

Question 28

# Metagenomics Advantages of Metagenomics

Answer 28

More comprehensive than 16S rRNA sequencing. Reveals functional genes (e.g., antibiotic resistance).

Question 29

# Metagenomics Limitations of Metagenomics

Answer 29

Expensive & computationally intensive.

Question 30

# Applications of 16S rRNA Gene Sequencing & Metagenomics Human Gut Microbiome Studies

Answer 30

16S rRNA: Identified dominant bacterial groups (Bacteroidetes & Firmicutes). Metagenomics: Revealed functional genes (e.g., metabolism, antibiotic resistance).

Question 31

# Applications of 16S rRNA Gene Sequencing & Metagenomics Marine Microbial Communities

Answer 31

Metagenomics uncovered previously uncultured microbes & their roles in biogeochemical cycles.

Question 32

# Applications of 16S rRNA Gene Sequencing & Metagenomics Antibiotic Resistance in Hospitals

Answer 32

Metagenomics identified resistance genes and their potential spread among pathogens.