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Walk through of sequencing process

A brief introduction to sequencing methodologies
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Hello, everyone. Welcome to this tutorial. Here, we are going to have a walkthrough of sequencing where we shall explore Sanger sequencing and Next-Generation Sequencing (NGS). Sanger sequencing. It was first developed in the 1970s by Sir Frederick Sanger. It is the gold standard of sequencing. It is still used today for routine applications and to validate NGS data. Sanger sequencing is also known as sequencing by chain termination. It uses chain-terminating dideoxyribonucleotide Triphosphates, ddNTPs, that are tagged with fluorochromes. A ddNTP is a deoxyribonucleotide Triphosphate, dNTP, without the free 3 prime OH group. This means that they are incapable of chain extension in case they are inserted into a growing chain by DNA polymerase.
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In classical Sanger sequencing, Sanger set up four different reactions, with each having all the basic PCR components, including a primer, DNA template, a polymerase, dNTPs. He also added ddNTPs in very small amounts. For more than Sanger sequencing, all these reactions are combined and run as a multiplex. The resulting reaction produces extension products of varying length, terminated by ddNTPs at the 3 prime end. These fragments are then separated by capillary electrophoresis. The extension products are injected by an electrical field or current into a capillary tube filled with a gel polymer. The extension products move from the negatively charged end to the positively charged end, and the speed at which they move is inversely proportional to their molecular weight.
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This process separates the extension products by size at a resolution of one base. A laser excites the dye-labelled ends of the fragments as they pass through the capillary tube. The excited dye emits a signature light that is detected by the sensor. The detected light is translated into a base call by a programme. At the end of the process, the Sanger sequencer gives a series of chromatographs together with the called bases as a single .ab1 file. Next-generation sequencing. Also known as massively parallel sequencing, is a powerful technique that has enabled advances in personalised medicine, genetic disease research, and clinical diagnosis. Examples of next-generation sequencing platforms include the MiSeq, the HiSeq, 454, SOLiD, ion proton.
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Although many NGS technologies have been recently developed over the years, they all share the same common features, and these include the nucleic acid extraction, sample preparation, sequencing, and data analysis. On nucleic acid extraction, cells, viruses are lysed to release the DNA and RNA. The RNA or DNA is then purified by various methods and kits. The RNA or DNA is then eluted and concentrated. Quality control of the extracted DNA or RNA is crucial. Sample preparation. The goal is to add sequencing adaptors to the DNA or RNA to be sequenced. It starts with target selection, where DNA or RNA are subjected to fragmentation or PCR. Short oligo motifs, also known as adapters, are added to either ends of each fragments or amplicons.
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The end product of sample preparation is what we call a sequencing library, where all fragments or amplicons are tagged with sequencing adapters. The library is then quality controlled and loaded onto the sequencer. Sequencing. The DNA with adapters is fast amplified into clones or clusters. After a series of chemical reactions that either produce change in pH, luminescence, or fluorescence, signals are captured by cameras and computers, which are then translated into base calls. Data analysis. Data from the sequencer contains the base calls and their quality scores. This is quality controlled. The data is then aligned or assembled de novo to generate consensus sequences that are used to answer biological questions.

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.

This is an additional video, hosted on YouTube.

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.

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