Biochemistry Questions Biochemistry Questions / Compare and contrast the different generations of sequencing technologies, including Sanger sequencing, Next Generation sequencing, and current generation sequencing. Discuss the advantages and disadvantages of each.

There are many sequencing technologies available now, each with their own advantages and issues.

Sanger sequencing, first developed by Fred Sanger, was the first sequencing technology to be developed, and is still dominant. It allows for relatively long read lengths, and has a reasonable accuracy. This technology is affordable, allowing it to be used in many laboratories, with a major use being the human genome project.

The development of next generation sequencing (NGS) was prompted by the human genome project, making it easier to sequence long genomes, as well as reducing cost and complexity. By carrying out thousands of simultaneous Sanger sequencing reactions, it is possible to increase speed, as well as allowing multiple different genomes to be sequenced simultaneously. This takes a shotgun approach, with the final consensus sequence being assembled computationally. Illumina is the dominant NGS technology, and is widely used.

Third, or current, generation sequencing technologies are still being developed. The two dominant forms are Nanopore and PacBio, both with good accuracy, very long sequence reads (up to the low megabases), and reduced costs. These also reduce the preparation required for sequencing. In NGS, Illumina sequencing requires the addition of adaptor molecules, and the breaking down of DNA strands to short sections around 300bp long. This makes it more time consuming and costly, due to the consumables (which also includes a flow cell and reversible dye-terminator nucleotides) being proprietary. Nanopore sequencing involves pulling the DNA or RNA strand through a pore protein (CsgG), and recording the flow of electrons around the nucleic acid strand. This has allowed for very long read lengths, while having no preparation steps - sequencing can be done directly from biological samples. PacBio sequencing uses fluorescent nucleotides and a polymerase immobilised in a zero-mode waveguide, allowing for long read lengths, as well as high accuracy. Consensus sequences can be generated easily through the use of SMRT-bell adaptors, circularising the DNA molecule and allowing for continuous replication displacing an existing strand. Both Nanopore and PacBio sequencing were used in the telomere-to-telomere project (2022), allowing the complete sequencing of every base in the human genome. This was previously not possible with Sanger and NGS technologies, as the short read lengths were not able to resolve long repeat regions.

Third generation sequencing technologies are becoming more prevalent, with improved accuracy and reduced cost, allowing for faster, more accurate, and cheaper DNA sequencing, improving accessibility and allowing both genomics and bioinformatics to advance. This could allow for improved drug usage in the future, where DNA mutations are analysed to determine which treatment options would be best, or reducing the risk of antimicrobial resistance by testing the bacteria first to determine its susceptibilities.