The 3 generations of DNA sequencing

What are the 3 generations of DNA sequencing?

1. First generation → Sanger sequencing

2. Second generation → Next-Generation Sequencing (NGS), such as Illumina

3. Third generation → Long-read sequencing, such as Oxford Nanopore and PacBio

What is Sanger sequencing?

A first-generation sequencing method and a "gold standard" for small-scale DNA sequencing/verification.

What reagents are needed for Sanger sequencing?

• DNA primer

• DNA polymerase

• Normal dNTPs

• Special ddNTPs

What is the main idea behind Sanger sequencing?

Chain termination — ddNTPs stop DNA synthesis due to a missing extra hydroxyl (-OH) group

What is different about ddNTPs?

ddNTPs are missing the 3′-OH group.

Why does missing the 3′-OH stop DNA synthesis?

DNA polymerase needs the 3′-OH to form the next phosphodiester bond. Without it, no more nucleotides can be added.

What happens when a ddNTP is incorporated?

The DNA strand stops growing.

What does Sanger produce?

DNA fragments of different lengths, each ending in a ddNTP.

How are the Sanger fragments analyzed?

They are separated by size, usually using electrophoresis.

How do you know which base is at the end of each fragment?

Each ddNTP has a fluorescent label/color that identifies whether the terminal base is A, T, G, or C.

What is second-generation sequencing?

Next-generation sequencing (NGS), such as Illumina sequencing.

What is the major advantage of NGS?

It can sequence millions of DNA molecules at the same time (in parallel).

Where does Illumina sequencing occur?

On a flow cell.

What happens during library preparation?

DNA is cut/sheared into smaller fragments, typically around 200–400 bp, and adapters are attached to the ends.

Why are adapters added?

They allow the DNA fragments to attach to the flow cell and participate in sequencing.

What is bridge amplification?

A process that makes thousands of identical copies of each DNA fragment in one location.

Why is bridge amplification needed?

It creates a cluster with enough DNA copies to produce a strong signal that the instrument's camera can detect.

What is a cluster?

A spot on the flow cell containing many identical copies of the same DNA fragment.

What is sequencing by synthesis (SBS)?

DNA is sequenced by building the new DNA strand one nucleotide at a time.

What happens during SBS?

Fluorescently labeled nucleotides are added, and the instrument detects the color from each cluster.

What is a "base call"?

Determining which base (A, T, G, or C) was added based on its detected signal/color.

What is third-generation sequencing?

Sequencing technologies that can read individual DNA molecules, often producing very long reads, without bridge amplification.

What is Bridge amplification?

Bridge amplification = making lots of copies of one DNA molecule so the sequencing machine can detect its signal.

What is a major difference from Illumina?

Third-generation methods generally do not require bridge amplification and can sequence single molecules in real time.

What is nanopore sequencing?

A sequencing method that passes DNA through a tiny protein pore and detects changes in electrical current.

What does a motor protein do?

It helps feed/control the DNA through the pore.

What is actually measured in nanopore sequencing?

Changes in ionic current as different DNA bases pass through the pore.

What is a major strength of nanopore sequencing?

It can produce ultra-long reads.

What is another advantage of nanopore sequencing?

Some devices are portable, including USB-sized sequencing devices.

What is the main trade-off between short-read and long-read DNA sequencing?

Short read: Cheap, high coverage, accurate, but hard to process computationally.

Long read: Easier to assemble, great for repeated sequences thought, great for no-reference (De-novo) sequencing, But higher sequencing cost per Gb.