Genome Technology and Engineering

A genome is the complete genetic information present in a cell of an organism.

In prokaryotes, the genome includes the DNA present in the nucleoid region and plasmids. In eukaryotes, the genome includes the DNA present in the chromosomes of the nucleus as well as in organelles such as mitochondria and plastids/chloroplasts.

So, the main difference is that prokaryotic genome is usually simpler and includes nucleoid DNA plus plasmids, while eukaryotic genome is distributed in the nucleus and organelles.

The chapter describes two types of genome mapping:

1. Genetic mapping
- It estimates distances between genetic loci by studying crossing over / recombination frequency.
- One map unit equals 1% observed crossover.
- Genetic distance is expressed in centimorgan (cM).
- It is useful for arranging loci according to inherited phenotype frequencies.
- Limitation: it depends on a limited number of known genetic loci, so the map is sparse and less useful for fine mapping.

2. Physical mapping
- It identifies actual positions on the genome using DNA-based markers.
- Map features include restriction sites, SSLPs, and STS.
- It gives a high-resolution map of the genome.
- It is more precise than genetic mapping because it is based on physical DNA features.

Comparative approach:
- Genetic mapping uses recombination frequency and shows relative genetic distances.
- Physical mapping uses DNA landmarks and shows actual physical locations.
- Genetic maps are coarser, whereas physical maps are higher resolution.
- Physical mapping is better for fine analysis of genome structure.

Digestion of DNA with BamHI is used in restriction mapping, which is a type of physical mapping.

In this method, restriction enzymes cut DNA at specific base sequences, and the resulting fragments are separated by agarose gel electrophoresis to make a map of restriction sites.

Application:
- It helps create a high-resolution physical map of the genome.
- It can be used to compare genomes and detect RFLP.
- In disease studies, comparison of restriction maps among diseased and healthy individuals may help identify a restriction site locus associated with the disease.

STS stands for Sequence Tagged Sites.

- They are unique DNA sequences of about 200–500 bp.
- Each occurs once in a genome and its location is known.
- Their presence or absence in a DNA fragment can be detected by PCR.

Role in genome mapping:
- STS act as landmarks on the genome.
- They help in creating a physical map.
- They are often combined with restriction maps to improve the resolution and effectiveness of genome mapping and association mapping.

The development of DNA sequencing technology has gone through three major stages:

1. First generation sequencing
- It was a multistep and laborious process.
- Chromosomes were separated by PFGE.
- DNA was digested with restriction enzymes and ligated to cloning vectors such as BAC, YAC, PAC.
- Clones were physically mapped and subcloned into sequencing plasmids.
- Sequencing used chain termination.
- Fragments were separated by capillary electrophoresis, and fluorescence peaks in a chromatogram showed the base sequence.
- It was accurate but time-consuming, labour-intensive, and expensive.

2. Next generation sequencing
- It uses massively parallel sequencing.
- Millions of reactions can occur in one machine.
- It avoids cloning and subcloning, so it is faster and cheaper.
- It gives high data accuracy due to high depth of coverage.

3. Third generation sequencing
- Uses nanopore sequencing.
- Measures changes in ionic current as single DNA bases pass through a nanopore.
- It is rapid, simple, low cost, and can produce very long reads.

Genomic workflow:
- DNA is obtained from the organism.
- It may be mapped or fragmented.
- Sequencing generates DNA reads.
- Computational analysis assembles and interprets the sequence.
- The final information is used for genome study, comparison, and application in biology and medicine.

Next generation DNA sequencing (NGS) has overcome the drawbacks of first-generation sequencing in several ways.

### How it improved over first generation
- First generation required cloning, subcloning, and many manual steps.
- It was time-consuming, labour-intensive, and costly.
- NGS uses a massively parallel approach, so millions of fragments are sequenced at once.
- It does not require time-consuming cloning and subcloning.
- It gives high data accuracy because of high depth of coverage.
- It has a broad range of applications.

### Methodology of Illumina NGS
1. DNA is fragmented into small pieces of about 1–2 kb.
2. Adaptor sequences are attached to the fragments.
3. The adaptors are complementary to oligonucleotides fixed on a flow cell.
4. DNA fragments attach to the flow cell.
5. Using Bridge PCR amplification, each fragment forms a bridge-like structure and is amplified into a cluster.
6. A sequencing primer binds to one adaptor.
7. Fluorescently tagged dNTPs are added.
8. Only one base is incorporated at a time.
9. The fluorescence is imaged to identify the added base.
10. The fluorescent tag is removed, and the next base is added.
11. This process is repeated to sequence millions of fragments in parallel.

### Limitation of NGS
- It generates short reads of about 75–300 bases, shorter than first-generation reads.

So, NGS is faster, cheaper, parallel, and more efficient, though it produces shorter sequence reads.

The unit of physical mapping is a physical distance, unlike the genetic map unit used in genetic mapping.

In the chapter, physical mapping is described as mapping based on actual DNA-based landmarks such as restriction sites, SSLP, and STS. It gives a high-resolution map of the genome.

### In detail
- Physical mapping identifies the specific locations on the genome.
- It uses DNA map features.
- Important physical map features are:
- Restriction enzyme sites for restriction mapping
- SSLPs such as microsatellites and minisatellites
- STS, unique DNA sequences with known locations
- These features help to arrange loci based on physical distances.

### Why it is important
- It provides a high-resolution map.
- It is useful for association mapping.
- It helps compare genomes and detect differences such as gain or loss of restriction sites.

So, the unit of physical mapping refers to the actual physical spacing of DNA loci on the chromosome, and the mapping is based on measurable DNA landmarks rather than recombination frequency.

Third generation sequencing technology is represented in the chapter by nanopore sequencing.

### Methodology
- DNA is first captured by a DNA helicase.
- The helicase unwinds double-stranded DNA.
- One strand is pushed through a nanopore formed by a porin-like protein on a synthetic membrane.
- An ionic current is maintained across the nanopore.
- As each base passes through the pore, it causes a specific change in current.
- These current changes are measured and used to identify the bases.

### Applications and advantages
- It provides rapid and simple sample processing.
- It can give sequencing results in real time.
- It is useful for genotyping, even in field settings.
- It produces very long reads, up to 1 Mb or more.
- It is relatively low cost.

So, nanopore sequencing is a fast, long-read, and practical third generation sequencing method.

The chapter mentions several variations of Next Generation Sequencing (NGS):

1. Whole Genome Sequencing (WGS)
- Determines the DNA sequence of the entire genome.
- Can be reference-based or de novo.

2. Targeted sequencing
- Sequences only selected genes or genomic regions.
- Used to detect mutations, insertions, and deletions.

3. Clinical exome sequencing
- Similar to targeted sequencing.
- Focuses on genes known to be disease-associated.
- More cost-effective than WGS.

4. ChipSeq
- Used to map genome-wide DNA binding sites of transcription regulators.

5. RNASeq
- Used to study the global gene expression profile or transcriptome.
- RNA is converted to cDNA and sequenced.

These are brief applications and forms of NGS described in the chapter.

Advanced sequencing technologies are used in many ways beyond simply reading DNA sequence.

### Applications mentioned in the chapter
- Whole Genome Sequencing (WGS):
- Determines the complete genome sequence.
- Useful for understanding inheritance, genetic disorders, and personalised disease treatment.
- Helps in selecting the best chemotherapy by reading cancer cell variation.

- Targeted sequencing:
- Detects variations in selected genomic regions.
- Useful for finding mutations, insertions, and deletions.

- Clinical exome sequencing:
- Helps in genetic diagnosis of disease-associated genes.
- Cost-effective compared to WGS.

- ChipSeq:
- Maps genome-wide DNA-binding sites of transcription regulators.
- Helps identify target genes regulated by transcription factors.

- RNASeq:
- Studies global gene expression or transcriptome.
- Helps quantify expression levels of genes.

- Metagenomics:
- Studies genomes of microbial communities.

- Nanopore sequencing:
- Useful for rapid genotyping and field-based testing.
- Produces long reads and real-time results.

So, advanced sequencing technologies are important in medicine, gene regulation studies, expression analysis, microbiology, and personalised treatment.

Metagenomics is the study of the total genetic material directly obtained from a microbial community without isolating or culturing the organisms individually.

### Applications
- Medical microbiology:
- Helps study microbes in the human body such as gut microbes and throat microbes.
- Agriculture:
- Useful for understanding microbial communities associated with soils and crops.
- Environmental microbiology:
- Helps study microbial diversity in different environments.
- Microbial diversity studies:
- Shows how microbial populations change when the environment changes.
- Discovery of novel genes and enzymes:
- Can identify enzymes with industrial use.
- Example: microbes from high-temperature sulphur springs may yield heat-resistant enzymes.
- Virus studies:
- Helps understand virus-host interaction, epidemiology, and evolution of viruses.
- Study of unusual environments:
- Can analyze microbes from places like a toilet seat or extreme environments.

### Special point
- Metagenomic samples contain multiple genomes, so data analysis is challenging and needs special computing algorithms.