Difference Between Nanopore and Illumina Sequencing

The key difference between nanopore and illumina sequencing is that nanopore sequencing is a third-generation sequencing technique that uses a nanopore to detect the sequence of a DNA molecule, while illumina sequencing is a second-generation sequencing technique that uses reversible dye terminators technology to detect the sequence of a DNA molecule.

DNA sequencing is the determination of a precise nucleotide or base sequence of a DNA molecule. There are many rapid methods to determine nucleic acid sequences that accelerate biological and medical research discoveries. One of the first DNA sequencing techniques (Sanger sequencing) was developed by Frederick Sanger in 1975 by adopting primer extension strategy at the MRC Centre, Cambridge, UK. Today, the majority of rapid DNA sequencing techniques belong to second-generation (next-generation) and third-generation DNA sequencing categories. Nanopore and illumina sequencing are two such new DNA technologies.

CONTENTS

1. Overview and Key Difference
2. What is Nanopore Sequencing
3. What is Illumina Sequencing
4. Similarities – Nanopore and Illumina Sequencing
5. Nanopore vs Illumina Sequencing in Tabular Form
6. Summary – Nanopore vs Illumina Sequencing

What is Nanopore Sequencing?

Nanopore sequencing is a third-generation sequencing technique that uses a protein nanopore to detect the nucleic acid sequence of a DNA molecule. In nanopore sequencing, the DNA passing through the nanopore changes its current. This change depends on the shape, size, and length of the DNA sequence. The resultant signal is decoded to get the specific DNA or RNA sequence. This method does not require modified nucleotides, and it performs in real-time.

Oxford Nanopore Technologies is a popular company that manufactures numerous nanopore sequencing devices. Most of the Oxford Nanopore sequencing devices have flow cells. This flow cell has a number of tiny nanopores that are embedded in electro resistant membrane. Each nanopore corresponds to its own electrode. This electrode connects to a channel and a sensor chip. This electrode measures the electric current flowing through the nanopore. When a molecule passes through a nanopore, its current changes or is disrupted. Moreover, this disruption produces a characteristic squiggle. This squiggle is then decoded to determine the DNA or RNA sequence in real-time.

What is Illumina Sequencing?

Illumina sequencing is a second-generation sequencing technique that uses reversible dye terminators technology to detect the sequence of DNA molecules. Solexa company, now a part of Illumina company, was founded in 1998.  This company invented this sequencing method based on reversible dye terminators technology and engineered polymerases.

In illumina sequencing method, the sample is first cleaved into short sections.  Therefore, in illumina sequencing, 100-150bp short reads or fragments are created at the beginning. These fragments are then ligated to generic adaptors and annealed to a slide. PCR is done to amplify each fragment. This creates a spot with many copies of the same fragment. Later, they are separated into single-stranded and subjected to sequencing.  The sequencing slide contains fluorescently labelled nucleotides, DNA polymerase, and a terminator. Because of the terminator, only one base is added at a time. Each cycle terminator is removed, and it allows the addition of the next base to the site. Furthermore, based on fluorescent signals, the computer detects the base added in each cycle. Illumina sequencing technology constructs the sequence within 4 to 56 hours.

What are the Similarities Between Nanopore and Illumina Sequencing?

• Nanopore and illumina sequencing are two sequencing techniques.
• Both are rapid and new sequencing methods.
• They are used to detect DNA and RNA sequences.
• Both have high accuracy.

What is the Difference Between Nanopore and Illumina Sequencing?

Nanopore sequencing is a third-generation sequencing technique that uses a nanopore to detect the sequence of DNA molecules. In contrast, illumina sequencing is a second-generation sequencing technique that uses reversible dye terminators technology to detect the sequence of DNA molecules. o, this is the key difference between nanopore and illumina sequencing. Moreover, nanopore sequencing has 92-97% accuracy, while illumina sequencing has 99% accuracy.

The following infographic lists the differences between nanopore and illumina sequencing in tabular form.

Summary – Nanopore vs Illumina Sequencing

High-throughput sequencing techniques include second-generation (short-read) and third-generation (long-read) sequencing methods. Nanopore and illumina sequencing are two new DNA technologies that belong to third-generation and second-generation (next-generation) DNA sequencing categories. Nanopore sequencing uses a nanopore to detect the sequence of DNA molecules. On the other hand, illumina sequencing uses reversible dye terminators technology to detect the sequence of DNA molecules. Thus, this is the summary of the difference between nanopore and illumina sequencing.

Reference:

1. “DNA Sequencing.” Oxford Nanopore Technologies.
2. “Illumina Dye Sequencing.” An Overview | ScienceDirect Topics.

Image Courtesy:

1. “Oxford nanopore MinION flow cell back” By Cirosantilli2 – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Illumina MiSeq sequencer” By Konrad Förstner – Own work (CC0) via Commons Wikimedia