DNA Sequencing
🧬 I. What is DNA Sequencing?
DNA sequencing is the process of determining the precise order of nucleotides (A, T, C, G) in
a DNA molecule.
Helps identify genes, mutations, and genetic variations
Foundation of genomics, personalized medicine, and evolutionary studies
🧪 II. Basic Principle of DNA Sequencing
DNA is made up of four nucleotides: Adenine (A), Thymine (T), Cytosine (C), Guanine (G)
DNA sequencing involves reading these bases in a specific order
Uses template DNA, primers, DNA polymerase, and nucleotides (regular and modified)
🔬 III. Methods of DNA Sequencing
1. Sanger Sequencing (Chain Termination Method)
Developed by Frederick Sanger in 1977
“First-generation” sequencing method
Steps:
1. DNA denaturation: Single-stranded template
2. Primer annealing: Binds to start of the sequence
3. Extension with:
Regular dNTPs
Fluorescently labeled ddNTPs (dideoxynucleotides), which terminate elongation
4. Capillary electrophoresis: Separates fragments by size
5. Laser detection: Reads fluorescent signals
✅ Pros:
High accuracy
Ideal for short DNA fragments (~500–1000 bp)
❌ Cons:
Time-consuming and expensive
Low throughput
2. Next-Generation Sequencing (NGS)
Also called high-throughput sequencing
Examples:
Illumina (Sequencing by Synthesis) – most common
Ion Torrent
454 Pyrosequencing (discontinued)
SOLiD sequencing
General Workflow:
1. Library preparation: DNA fragmented and adapters added
2. Amplification: Clonal amplification on flow cell
3. Sequencing:
Sequential addition of nucleotides
Signal (light or pH) is detected and recorded
4. Data analysis: Bioinformatics software interprets sequences
✅ Pros:
Massive parallel sequencing
High speed and throughput
Cost-effective for large genomes
❌ Cons:
Requires powerful computational analysis
Shorter read lengths than Sanger (but getting better)
3. Third-Generation Sequencing
Examples:
PacBio SMRT (Single-Molecule Real-Time)
Oxford Nanopore (MinION)
✅ Pros:
Long reads (10,000+ bp)
Real-time sequencing
Minimal sample prep
❌ Cons:
Higher error rates (being improved)
More expensive equipment (for some platforms)
📋 IV. Applications of DNA Sequencing
🧬 Clinical Diagnostics
Identifying mutations in genetic diseases (e.g., cystic fibrosis, BRCA1/2)
Pharmacogenomics (drug response based on genes)
Cancer genomics (tumor mutations, targeted therapy)
🦠 Infectious Disease Detection
Pathogen identification (e.g., TB, SARS-CoV-2)
Drug resistance mutations
🧫 Microbiology
16S rRNA sequencing for bacterial identification
Metagenomics (microbiome analysis)
🧠 Research & Genomics
Whole genome sequencing (WGS)
Whole exome sequencing (WES)
Transcriptome (RNA-seq)
Epigenomics and variation analysis (SNPs)
🧬 Forensics and Paternity
Short Tandem Repeat (STR) analysis
DNA fingerprinting
🧠 V. Interpretation of Results
Chromatogram (Sanger): Peaks for each nucleotide
FASTQ files (NGS): Sequence data with quality scores
Variant Calling: Detection of SNPs, indels, etc.
Alignment: Compare sequences to a reference genome
⚙️ VI. Advantages and Disadvantages
Method Advantages Limitations
Sanger Accurate, long Low throughput,
reads expensive
NGS (Illumina) Fast, high- Shorter reads,
throughput, cost- data analysis
efficient needed
Nanopore Long reads, real- Higher error rate
time, portable (improving)
(MinION)
🧾 VII. Summary Table
Sequenci Read Throughp Accuracy Main Use
ng Type Length ut Case
Sanger ~800– Low Very high Targeted
1000 bp genes
NGS 50–300 Very high High Genomes,
(Illumina) bp transcript
omes
Nanopore 10,000+ Moderate Moderate Long
bp –High –High reads,
field work
🔍 VIII. Common Terms to Know
Read: Sequence fragment output by the sequencer
Coverage (Depth): Number of times a region is read
Contig: Assembled continuous sequence
Reference genome: Known standard for alignment
Variant: Any change from the reference (SNP, deletion, etc.)
