Next Generation Sequencing

By Mark Comer

The development of Sanger sequencing in 1977 laid the foundation for DNA sequencing technologies and continued to be the leading technique for the discovery of DNA sequences until the 1990s.  With the genome of a bacteriophage the first to be sequenced in the same year, sequencing technology continued to be developed. Replacing the previously radioactive tags with fluorescent ones and the application of computer software increased the efficiency of sequencing. Despite all the success of Sanger sequencing and a variety of technological advances, it remained a laborious and time-consuming process. Most importantly, Sanger sequencing remained relatively low throughput which significantly restricted its applications. The birth of next generation sequencing began in earnest with Solexa and the Illumina corporation, although the less ubiquitous Oxford Nanopore and Pacific Bioscience’s bespoke platforms also offer significant advantages over Sanger sequencing. Third-generation sequencing techniques were built on a foundation of developments that occurred throughout the later part of the 1990s (Behjati S, Tarpey PS, 2013). The development of these technologies has catalysed a genomics revolution, pushed the frontiers in numerous fields, and has driven the increasing importance of bioinformatics approaches. Next generation sequencing approaches have also proved invaluable for public health, particularly for identification of novel SARS-CoV-2 variants.

The basis of Illumina’s ground-breaking sequencing-by-synthesis technology was rooted in a brainchild of David Klenerman and Shankar Balasubramanian at the University of Cambridge. But prior to this development, the first next generation sequencing technique known as pyrosequencing had been proposed in 1996. This method functioned by measuring luminescence produced as a result of pyrophosphate synthesis. This was the first truly high throughput sequencing method and the first advancement of sequencing beyond Sanger sequencing. Throughout the period of the later 1990s, the notion of a $1000 genome that could be sequenced within a day was an extremely radical idea. But through “massively-parallel” sequencing, this idea was soon realised (Goodwin S, McPherson JD, McCombie WR, 2016). The Illumina method revolves around fragmentation of sample DNA into small fragments followed by the ligation of adapter sequences. The adapter sequences allow for binding to a flow cell and aid in quality control, amplification by PCR leads to formation of distinct clusters of DNA in a synthetic flow cell composed of the same sequence. Utilising the same principles as Sanger sequencing, reversible chain-terminating nucleotides with fluorescent tags are used to provide a base call. Comparatively, this method allows for incredibly rapid sequencing. Perhaps, the most significant application of next generation sequencing is allowing easier and more rapid de novo assembly of a genome without a reference genome. Sequencing genomes again with next generation sequencing techniques also allows for the identification of novel variants and to fill in missing bases from a reference genome (Auton et al, 2015). This was most recently demonstrated with the human genome undergoing sequencing with Pacific Bioscience’s platform, this allowed for researchers to fill most of the gaps that remained in the reference genome that was first produced in 2003 (Reardon S, 2021). Next generation sequencing has also been combined with a variety of other technologies to allow for analysis of protein-DNA interactions with ChIP-seq, and analysis of the transcriptome was made possible by generating a cDNA library for RNA-seq.  Sanger sequencing does retain some usefulness, with a higher read length than the Illumina platform and comparatively better base call accuracy, it is preferred for sequencing of small regions in a few samples.

Oxford Nanopore and Pacific Bioscience next generation sequencing technologies differ significantly from Illumina’s sequencing by synthesis in several key ways. Oxford Nanopore’s platform is incredibly portable, the sequencing device is handheld and can be easily transported and boasts a higher read length than the Illumina platform. The portability of the device helps overcome the shortcomings of the Illumina method which cannot sequence genomes in the field or be deployed rapidly for use in public health initiatives especially for speedy detection of pathogens (Bowden et al, 2019) (Qin D, 2019) (Barzon et al, 2011). Moreover, the short read length of the Illumina method means that resolution of highly repetitive regions is low. The sequencing is made possible by observing changes in electrical current through a protein nanopore as the sample DNA or RNA is fed through the pore, with each nucleotide altering the current differently. This allows for real-time generation of the DNA-sequence. Another advantage of the platform is the ability to detect epigenetic changes through algorithmic methods, traditional assays do not allow for long sequencing reads due to damage to DNA. In short, the Oxford Nanopore platform allows for portable and rapid generation of nucleic acid sequences in real-time with a comparatively much higher read length than Illumina’s platform. Pacific Bioscience’s platform utilises immobilised polymerases, with the inclusion of fluorescent nucleotides and circularised sample DNA, base calls can be read in real-time like the Oxford Nanopore platform. The addition of each nucleotide results in a light ‘pulse’ that is detected and corresponds to the base call. The Pacific Biosciences platform is considered to be the first “third-generation sequencing platform” but is nonetheless relatively expensive and lower throughput in comparison to either Illumina or Oxford Nanopore (Rhoads et al, 2015).  Both platforms can allow for identification of structural variations and higher read lengths in comparison to the Illumina method.

In conclusion, the capability of next generation sequencing technologies has pushed the boundaries of biomedical science and spearheaded a greater understanding of the role of the genome in human health and disease. As technological advances continue, the accuracy, speed, and cost-effectiveness are likely to improve with new third-generation technologies allowing us unprecedented insights into the genome but also the transcriptome and epigenome.


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