DNA Sequencing Technologies - Next-Generation Sequencing

DNA sequencing times and costs have fallen dramatically over the past 20 years due to emergence of new sequencing technologies known as next-generation sequencing (NGS).

NGS methods are high-throughput, meaning they can sequence millions or even billions of DNA fragments at the same time, in parallel. NGS methods are also relatively much cheaper than traditional methods such as Sanger sequencing, meaning it is now feasible to sequence very large quantities of genome data.

There are many different next-generation DNA sequencing technologies, which use different methods to sequence DNA. Examples of widely used methods include Illumina sequencing and Nanopore sequencing.

 

Illumina

Illumina is a widely used NGS method, first available on a commercial scale in the early 2000s. It allows high numbers of short DNA strands to be sequenced simultaneously, using a sequencing-by-synthesis (SBS) approach.

  1. The target DNA sample for sequencing is cleaved into short sections, around 100 to 150 bases long.

 

  1. The DNA fragments are added to adaptors (very short chemically-synthesised DNA molecules), which are used to attach the DNA fragments to a flow cell (a special plate in which the sequencing process takes place).

 

  1. A reaction called ‘Bridge amplification PCR’ generates thousands of copies of each DNA fragment, forming clusters on the flow cell. In this reaction, a protein called DNA polymerase creates new DNA strands, using the fragments as a template.

 

  1. Bridge amplification PCR results in many clusters of DNA on the flow cell, each cluster consisting of thousands of copies of a given DNA fragment.

 

  1. Once clusters have been generated, repeated cycles of DNA synthesis begin, using the fragments as templates to build new DNA strands. These cycles are known as ‘chain-termination reactions’:
    • Modified nucleotides are added to the flow cell. These nucleotides are attached to a fluorescent dye, with each type of nucleotide (A, T, C, or G) dyed a different colour. The dye prevents any further nucleotides being added, so only one nucleotide is added to each strand per cycle.
    • In each cycle, one nucleotide is added to the new DNA strand that is being built. A camera then takes a picture of the flow cell, and a computer uses the colour of the dye to determine which nucleotide was added in that cycle to each cluster.
    • The dye is then removed, so further nucleotides can be added to the growing sequence, and another cycle starts.
    • This process repeats until the full strand of each DNA fragment has been sequenced.

Illumina sequencing - adaptor binding
Illumina sequencing - bridge amplification PCR
Illumina sequencing - DNA clusters
Sanger sequencing - chain termination reactions
Illumina sequencing - flow cell imaging

Nanopore sequencing

Nanopore sequencing was first released commercially in 2014. It has transformed genomic research because it can sequence very long DNA strands over a million bases in length in one go, meaning sequences no longer need to be assembled by a computer from fragments of only a few hundred bases long. The technology is also available as portable devices, so sequencing can be carried out in remote areas with limited laboratory resources for the first time.

 

  1. An extremely small hole (known as a nanopore) is embedded in a membrane separating two chambers, and an electric current is applied across the membrane
Nanopore sequencing - Nanopore set-up

 

  1. The DNA molecule is squeezed through the nanopore, one nucleotide at a time

 

  1. Each different nucleotide causes tiny changes to the electric current, which are detected by a computer and translated to give the sequence of the nucleotides
Nanopore sequencing - nucleotide detection

 

 

 

10 August 2023
Jemima Swain

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