Microbial Genomics and Genetic Manipulation: Whole genome sequencing and annotation of microbial genomes

Microbial Genomics and Genetic Manipulation:
Whole genome sequencing and annotation of microbial genomes

1. Unlocking Microbial Secrets:
Whole Genome Sequencing
and Annotation
Welcome to the forefront of microbiology. This presentation guides you
through the process and profound applications of Whole Genome
Sequencing (WGS) and annotation in the microbial world.

2. Why Microbial Genomics Matters: A Modern
Revolution
Deep Insight
Go beyond basic identification to
understand an organism's full
functional potential.
Precision
Enable highly detailed tracking
of outbreaks and evolutionary
changes with genomic data.
Innovation
Accelerate drug discovery,
diagnostics development, and
synthetic biology applications.
Microbial genomics provides the foundational blueprint for tackling global health challenges, from pandemics to drug
resistance.

3. The Journey Begins: From Microbe to DNA
1. Sample Acquisition
Starting with a pure culture of the microbe from an environmental
or clinical source.
2. DNA Extraction
Careful lysis of the cell wall to isolate high-quality, high-
molecular-weight genomic DNA.
3. Quality Control
Checking DNA concentration and purity using spectrophotometry
or fluorometry to ensure optimal sequencing results.
4. Library Preparation
Fragmenting the DNA and adding specific adapter sequences
required for the chosen sequencing platform.

4. Sequencing Technologies: Peeking into the Genetic Code
The choice of sequencing technology is crucial, depending on the required read length, throughput, and accuracy.
Sanger Sequencing (First Generation)
Gold standard for accuracy, but low-throughput.
Used primarily for validation or sequencing short
fragments/plasmids.
PacBio HiFi (Third Generation)
Produces highly accurate long reads (HiFi reads).
Excellent for assembling complex or repetitive microbial
genomes.

5. Illumina & Nanopore: High-Throughput & Real-Time
Insights
Illumina (Second Generation)
Known for massive throughput and high accuracy through
short reads (150-300 bp). Ideal for deep coverage,
detecting rare variants, and large-scale population studies.
Benefit: Lowest cost per base.
Drawback: Difficulty with highly repetitive regions due
to short read lengths.
Oxford Nanopore Technologies (ONT)
Provides ultra-long reads (up to Mb) in real-time, using a
portable, miniature device. Essential for rapid field
diagnostics and real-time outbreak monitoring.
Benefit: Long reads simplify assembly; portability for
on-site sequencing.
Drawback: Historically lower initial accuracy compared
to Illumina, though improving rapidly.

6. Assembling the Puzzle: From Reads to a
Complete Genome
1. Contigs and Scaffolds
Sequencing machines produce millions of short
or long "reads." Assembly is the computational
process of overlapping these reads to form
continuous segments called contigs.
Contigs are then ordered and oriented into
scaffolds using paired-end data or long reads,
bridging the gaps between them.
2. Genome Finishing
This stage involves closing any remaining gaps
in the scaffold using targeted sequencing or
manual curation, aiming for a single, complete,
circular chromosome for the microbe.
Hybrid assembly combining high-
accuracy short reads (Illumina) with
structure-providing long reads
(Nanopore/PacBio) is now the
preferred method for high-quality
microbial WGS.

7. Unveiling the Blueprint: Gene Prediction and Functional Annotation
Functional AnnotationGene PredictionGenome Assembly
1 Gene Prediction
Identifying regions of the DNA sequence that encode
proteins, tRNAs, or rRNAs. Computational tools search
for start/stop codons and ribosome binding sites (Open
Reading Frames - ORFs).
2 Functional Annotation
Assigning biological meaning to the predicted genes. This
involves comparing the predicted protein sequences
against extensive public databases like GenBank, UniProt,
and KEGG.
3 Metabolic Pathway Reconstruction
Mapping annotated genes to complete metabolic
pathways to understand how the microbe generates
energy, synthesises compounds, and interacts with its
environment.

8. Beyond the Sequence: Applications in Public Health
Pathogen Identification
Rapidly and accurately identifying
causative agents in clinical samples,
including previously unknown or difficult-
to-culture organisms.
Antimicrobial Resistance (AMR)
Pinpointing the exact genes and
mechanisms responsible for antibiotic
resistance, guiding more effective
treatment strategies.
Outbreak Surveillance
Real-time genomic tracking to trace the
source, transmission routes, and
evolution of infectious disease outbreaks
globally.

9. Battling Resistance & Tracing Evolution: Real-World Impacts
Genomics in Action: AMR Surveillance
WGS allows health authorities to monitor the spread of multi-drug resistant (MDR) "superbugs" in hospitals and communities. By
comparing genomes, they can identify emerging resistance mechanisms and clone lineages before they become widespread.
Evolutionary Studies
By comparing the genomes of related microbial strains, researchers can build phylogenetic trees to understand adaptation, host-
switching, and diversification over time. This is critical for vaccine development and understanding virulence factors.

10. Your Guide to the Microbial Universe: Key
Takeaways
WGS is Foundational
It provides the ultimate resolution for studying
microbes, replacing traditional, lower-resolution
methods.
Technology Convergence
Hybrid assembly (combining short and long reads)
delivers the most complete and accurate microbial
genome maps.
Annotation is Interpretation
Genome assembly is just data; annotation provides the
essential biological context (what the genes do).
Impact on Public Health
Applications in AMR, outbreak tracing, and evolutionary
understanding are transforming modern medicine and
epidemiology.

11. Thank you