Summary of Bacterial and Viral Genomics
Bacterial and Viral Genomics: A Comprehensive Student Guide
Introduction
Bacterial genomics methods & sequencing cover the laboratory and computational approaches used to read, assemble, and interpret bacterial genomes. This material explains major sequencing technologies, sample workflows, assembly strategies, and practical considerations for bacterial genome projects. It is designed for self-study and focuses on methods rather than evolutionary, pandemic, host–pathogen, or viral topics.
Definition: Genomics is the study of whole genomes of organisms, combining molecular biology and bioinformatics to determine sequence, structure, and function.
1. Overview of a bacterial genome project
- Plan the project: define goals (complete genome, draft assembly, plasmid detection, methylation mapping).
- Prepare DNA: high-quality, high-molecular-weight DNA is often required for long-read platforms.
- Choose sequencing strategy: short reads, long reads, or hybrid approaches (see table below).
- Library preparation: fragmentation, adapter ligation, optionally size selection.
- Sequencing run and primary basecalling.
- Bioinformatic processing: quality control, assembly, polishing, annotation, and downstream analyses.
Definition: Assembly is the computational process of reconstructing the original genome sequence from sequencing reads.
2. Sequencing technologies — key characteristics
Short-read sequencing (Illumina)
- Chemistry: sequencing-by-synthesis with reversible terminators
- Typical read length: $36$–$250$ bp
- Strengths: very high per-base accuracy, high throughput, low cost per base
- Limitations: short reads complicate assembly across repeats and structural variants
Long-read sequencing (PacBio, Oxford Nanopore)
- PacBio (SMRT): read lengths typically $250$–$10{,}000$ bp (can be longer); can detect modified bases by kinetic signal
- Oxford Nanopore: read lengths up to $>4{,}000{,}000$ bp reported; real-time sequencing, portable devices (MinION)
- Strengths: resolve repeats, plasmids, genome structure, detect modifications, enable near-complete assemblies
- Limitations: higher raw error rates historically (improving rapidly), needs high-quality DNA for longest reads
Sanger sequencing (dideoxy)
- Read length: $700$–$900$ bp
- Use: validation, finishing, small-scale projects
- Strengths: very accurate per-read
- Limitations: low throughput and high cost per base
💡 Did you know?Fun fact: Nanopore sequencing has produced reads over 4 million bases in length, enabling assembly across entire bacterial chromosomes in a single read for some samples.
3. Comparing platforms (summary table)
| Platform | Read length (bp) | Throughput | Best uses |
|---|---|---|---|
| Sanger | $700$–$900$ | Low | Validation, finishing |
| Illumina (MiSeq/HiSeq) | $36$–$250$ | High | Resequencing, variant calling, short-read assemblies |
| PacBio (SMRT) | $250$–$10{,}000$ | Moderate | Structural resolution, methylation detection, de novo assembly |
| Oxford Nanopore (MinION/GridION) | $10{,}000$–$50{,}000+$ | Variable (real-time) | Long-range assembly, metagenomics, field sequencing |
4. Typical workflows for bacterial genomes
A. Short-read-only assembly (Illumina)
- Fragment genomic DNA and prepare a paired-end library.
- Sequence to high coverage (e.g., $50$–$200\times$).
- QC reads (trim adapters, filter low-quality bases).
- Assemble with de Bruijn graph assemblers (e.g., SPAdes).
- Result: high base accuracy contigs, possible fragmentation at repeats.
B. Long-read-only assembly (Nanopore or PacBio)
- Extract high-molecular-weight DNA, prepare long-read library.
- Sequence to moderate coverage (e.g., $30$–$100\times$ depending on platform).
- Assemble with overlap-layout-consensus (OLC) or long-read assemblers (e.g., Flye, Canu).
- Polish assembly: use long-read polishing tools, optionally use short reads for final polishing.
- Result: contiguous assemblies often including closed chromosomes and plasmids.
C. Hybrid assembly (short + long reads)
- Combine short-read da
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Bacterial Genomics Methods
Klíčová slova: Bacterial genomics methods & sequencing, Bacterial genomics evolutionary studies, Historic pandemics, Bacterial pathogen genomics, Host–pathogen genetics, Viral genomics
Klíčové pojmy: Define project goals before sequencing, High-molecular-weight DNA improves long-read results, Use short reads for high base accuracy and long reads for contiguity, Hybrid assembly (long + short reads) often yields closed genomes, Polish assemblies with appropriate tools (long- and short-read polishers), Evaluate assemblies with N50, contig count, and completeness tools (BUSCO/CheckM), Long reads enable detection of base modifications (e.g., $\mathrm{m}^6\mathrm{A}$), Nanopore provides real-time, ultra-long reads suitable for plasmid resolution, Sanger sequencing remains useful for validation/finishing, Aim for $>50\times$ short-read or $30$–$100\times$ long-read coverage for bacterial genomes
## Introduction
Bacterial genomics methods & sequencing cover the laboratory and computational approaches used to read, assemble, and interpret bacterial genomes. This material explains major sequencing technologies, sample workflows, assembly strategies, and practical considerations for bacterial genome projects. It is designed for self-study and focuses on methods rather than evolutionary, pandemic, host–pathogen, or viral topics.
> Definition: Genomics is the study of whole genomes of organisms, combining molecular biology and bioinformatics to determine sequence, structure, and function.
## 1. Overview of a bacterial genome project
1. Plan the project: define goals (complete genome, draft assembly, plasmid detection, methylation mapping).
2. Prepare DNA: high-quality, high-molecular-weight DNA is often required for long-read platforms.
3. Choose sequencing strategy: short reads, long reads, or hybrid approaches (see table below).
4. Library preparation: fragmentation, adapter ligation, optionally size selection.
5. Sequencing run and primary basecalling.
6. Bioinformatic processing: quality control, assembly, polishing, annotation, and downstream analyses.
> Definition: Assembly is the computational process of reconstructing the original genome sequence from sequencing reads.
## 2. Sequencing technologies — key characteristics
### Short-read sequencing (Illumina)
- Chemistry: sequencing-by-synthesis with reversible terminators
- Typical read length: $36$–$250$ bp
- Strengths: very high per-base accuracy, high throughput, low cost per base
- Limitations: short reads complicate assembly across repeats and structural variants
### Long-read sequencing (PacBio, Oxford Nanopore)
- PacBio (SMRT): read lengths typically $250$–$10{,}000$ bp (can be longer); can detect modified bases by kinetic signal
- Oxford Nanopore: read lengths up to $>4{,}000{,}000$ bp reported; real-time sequencing, portable devices (MinION)
- Strengths: resolve repeats, plasmids, genome structure, detect modifications, enable near-complete assemblies
- Limitations: higher raw error rates historically (improving rapidly), needs high-quality DNA for longest reads
### Sanger sequencing (dideoxy)
- Read length: $700$–$900$ bp
- Use: validation, finishing, small-scale projects
- Strengths: very accurate per-read
- Limitations: low throughput and high cost per base
Fun fact: Nanopore sequencing has produced reads over 4 million bases in length, enabling assembly across entire bacterial chromosomes in a single read for some samples.
## 3. Comparing platforms (summary table)
| Platform | Read length (bp) | Throughput | Best uses |
|---|---:|---:|---|
| Sanger | $700$–$900$ | Low | Validation, finishing |
| Illumina (MiSeq/HiSeq) | $36$–$250$ | High | Resequencing, variant calling, short-read assemblies |
| PacBio (SMRT) | $250$–$10{,}000$ | Moderate | Structural resolution, methylation detection, de novo assembly |
| Oxford Nanopore (MinION/GridION) | $10{,}000$–$50{,}000+$ | Variable (real-time) | Long-range assembly, metagenomics, field sequencing |
## 4. Typical workflows for bacterial genomes
### A. Short-read-only assembly (Illumina)
- Fragment genomic DNA and prepare a paired-end library.
- Sequence to high coverage (e.g., $50$–$200\times$).
- QC reads (trim adapters, filter low-quality bases).
- Assemble with de Bruijn graph assemblers (e.g., SPAdes).
- Result: high base accuracy contigs, possible fragmentation at repeats.
### B. Long-read-only assembly (Nanopore or PacBio)
- Extract high-molecular-weight DNA, prepare long-read library.
- Sequence to moderate coverage (e.g., $30$–$100\times$ depending on platform).
- Assemble with overlap-layout-consensus (OLC) or long-read assemblers (e.g., Flye, Canu).
- Polish assembly: use long-read polishing tools, optionally use short reads for final polishing.
- Result: contiguous assemblies often including closed chromosomes and plasmids.
### C. Hybrid assembly (short + long reads)
- Combine short-read da