DNA sequencing techniques (Sanger, NGS, human genome sequencing)

Introduction

DNA sequencing is the process of determining the precise order of nucleotides (adenine, thymine, cytosine, and guanine) within a DNA molecule. It has revolutionized modern biology and medicine, enabling groundbreaking advances in genomics, diagnostics, and personalized medicine. This article explores the main sequencing techniques— Sanger sequencing, Next-Generation Sequencing (NGS), and their roles in Human Genome Sequencing.

History of sequencing

1. Sanger sequencing (first generation sequencing)

Developed by: Frederick Sanger in 1977

Principle

Sanger sequencing uses dideoxynucleotide triphosphates (ddNTPs) that terminate DNA strand elongation. DNA polymerase synthesizes new strands of DNA from a template strand, but when a ddNTP is incorporated, the elongation halts due to the absence of a 3’-OH group.

Each of the four reactions contains all four dNTPs and a small amount of one of the four ddNTPs (ddATP, ddTTP, ddCTP, or ddGTP), labeled with a fluorescent tag. After the synthesis, the fragments are separated by capillary electrophoresis, and the resulting fluorescent signals are read to determine the DNA sequence.

Applications

• Small-scale sequencing (single genes or small genomes)
• Mutation detection
• Validating NGS results

Advantages

• High accuracy (~99.99%)
• Long read lengths (~700–1000 bp)

Limitations

• Low throughput
• Expensive and time-consuming for large-scale projects

Link for a video explanation for those who need a visual input:

https://youtu.be/X9566yI2cBo?si=9AkDFBcVY_BhHub4

2. Next-generation sequencing

Introduced: Early 2000s

Also known as: Second-generation sequencing

Overview

NGS refers to high-throughput sequencing technologies that can sequence millions of DNA fragments in parallel. Unlike Sanger sequencing, NGS is massively parallel and allows whole genomes to be sequenced quickly and affordably.

Common Platforms

• Illumina (Sequencing by synthesis)
• Ion Torrent (Semiconductor sequencing)
• Roche 454 (Pyrosequencing – now discontinued)
• SOLiD (Sequencing by oligonucleotide ligation – discontinued)

Principle (Illumina as example)

1. Library Preparation: DNA is fragmented, and adapters are added.
2. Cluster Generation: Fragments bind to a flow cell and amplify to form clusters.
3. Sequencing: Fluorescently labeled nucleotides are added one by one, and a camera captures the emission after each incorporation.
4. Data Analysis: Software assembles reads and aligns them to a reference genome.

Applications

• Whole-genome sequencing (WGS)
• Whole-exome sequencing (WES)
• RNA sequencing (transcriptomics)
• Epigenetics (methylation analysis)
• Microbiome studies

Advantages

• High throughput
• Low cost per base
• Fast and scalable

Limitations

• Short read lengths (usually 150–300 bp)
• Complex bioinformatics required
• Higher error rates compared to Sanger

Learn by watching a video:

https://youtu.be/WKAUtJQ69n8?si=FjzUzRULCsQWS0dn

3. Human genome sequencing

Human Genome Project (HGP)

• Duration: 1990–2003, in 2022 ( sequencing became complete)
• Goal: Sequence the entire human genome (~3 billion base pairs)
• Method Used: Sanger sequencing
• Outcome: First full sequence of the human genome
• Cost: ~$2.7 billion

Modern Genome Sequencing

With NGS, whole human genomes can now be sequenced within days at a fraction of the cost (less than $1000 in many cases). Projects like the 1000 Genomes Project, UK Biobank, and All of Us (USA) have relied on NGS to study population-level genomic data.

Emerging Technologies

• Third-Generation Sequencing (TGS): Single-molecule real-time sequencing (SMRT, e.g., PacBio) and nanopore sequencing (e.g., Oxford Nanopore) allow long-read sequencing up to 2 million bp.
• Advantages of TGS: Detection of structural variants, better genome assembly, epigenetic analysis.

Impact on Medicine

• Cancer genomics (targeted therapies)
• Inherited disease diagnosis (e.g., cystic fibrosis, BRCA1/2 mutations)
• Pharmacogenomics
• Prenatal testing (non-invasive prenatal testing, NIPT)

Learn by watching:

https://youtu.be/-hryHoTIHak?si=EPKACvGBeERLEfbN

Reference

Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74(12):5463-5467. PMID: 271968

Metzker ML. Sequencing technologies—the next generation. Nat Rev Genet. 2010 Jan;11(1):31-46. doi:10.1038/nrg2626

International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860-921. DOI