Walk through of sequencing process

This animation briefly introduces the steps for sequencing methodologies currently in use. The first part outlines the dye-terminating sequencing, also known as Sanger sequencing. The second part describes a general protocol of the Next-Generation sequencing method. This video was created to showcase the techniques currently being used for SARS-CoV-2 genome sequencing in Uganda and many other countries. The video focuses on Illumina technology.

The emergence of the genomic era was fueled by DNA sequencing techniques developed by several scientists in the 1970’s including Fred Sanger’s influential method, which is still used nowadays in research and diagnostic labs. This technique uses a DNA polymerase and chain-terminating nucleotides to identify up to 1000bp DNA sequences in less than 2 hours. It has a high cost per base when compared to other technologies.

A new group of sequencing platforms, known as second-generation sequencing, started emerging in the 2000s, characterised by parallel sequencing of hundreds of thousands to billions of DNA fragments- this made sequencing at a large scale faster and cheaper. Illumina and Ion torrent are two of the current platforms and emerged in 2006 and 2010, respectively. They both require a PCR step, whereby each individual DNA fragment is amplified to generate a cluster of identical sequences. Clusters act as individual sequencing reactions inside the sequencing chip. They both use a polymerase to sequence the DNA fragments but Illumina uses fluorescently labelled nucleotides whereas Ion Torrent measures pH changes. They are both characterised by short read lengths (75-300bp reads with Illumina and 200-400bp reads with IonTorrent) but IonTorrent has shorter running times (2 hours compared to up to 56 hours with Illumina) and lower equipment costs, whereas Illumina is characterised by a lower error rate and lower cost per base.

Third-generation sequencing technologies such as PacBio and Oxford Nanopore Technologies (ONT) introduced in the 2010s, support longer read lengths. As opposed to second-generation sequencing platforms, they don’t require a PCR step to generate clusters and they enable real-time data analysis. ONT uses nanopores and a detector that measures changes in current to identify nucleotide stretches and has a high error rate at the single read level. PacBio uses a polymerase fixed in a chamber and fluorescently labelled nucleotides- it has the option to use circularised fragments to increase accuracy. The sequencing run time varies with both platforms but can be very short, even one hour can be enough to generate the required data. ONT’s unique features, including the option to use portable sequencers, greatly contributed to its dissemination across different areas of research.

Globally, many sequencing laboratories use nanopore sequencing techniques to facilitate viral genomic surveillance. The additional portability of these systems means that additional sequencing capacity can be enabled quickly and with minimal existing infrastructure needs.

To learn more about Oxford Nanopore Technologies, please watch the video below.

You can find detailed information about sequencing technologies for download below.

We will cover details of sequencing technologies in our Sequencing course to be launched in September 2022.