DNA sequencing: the next generation
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Although Sanger sequencing was good, it was still quite slow and cumbersome for a world that was now itching to Sequence All The Things. Scientists knew that if they wanted to take a leap forward in the speed of DNA sequencing, they needed to stop relying on synthesising DNA fragments and then looking at them afterwards, and start analysing the DNA synthesis process in real-time, reading off each letter directly as it was added.
In 1996, Mostafa Ronaghi, Mathias Uhlen and Pȧl Nyŕen from the Royal Institute of Technology in Stockholm introduced pyrosequencing, the first real-time DNA sequencing platform.
In pyrosequencing, single DNA strands are attached to tiny beads in individual wells and nucleotides are introduced one at a time, together with DNA polymerase.
If the correct nucleotide is introduced and is added to the strand, a chain of chemical reactions is initiated, resulting in the emission of light. The remaining nucleotides are washed away before the next is added, and the process is repeated. Detecting these light emissions after adding a known nucleotide allows scientists to work out the sequence of bases in the entire DNA strand.
So, for example, you add a bunch of As - no flash. Wash them out and add Ts - no flash. Wash and add Gs - flash, so you know the first letter in the sequence must be a G. Then you start the whole cycle again to discover the next letter.
It sounds like a bit of a faff, but by this point automation was making pyrosequencing possible at scale. Ronaghi, Uhlen and Nyren licensed their technology to 454 life sciences, and in 2005 it became the first commercially available next-generation sequencing method.
The platform represented a massive leap forward in better speed at lower cost by allowing large numbers of DNA fragments to be analysed at the same time, known as mass parallel sequencing. The 454 machines were able to generate reads of around 400 to 500 letters from a million or so beads at the same time, representing a huge boost in capacity. But just as the Sanger method emerged from under the nose of Maxam and Gilbert, there was a challenger just around the corner.
Following the success of 454, other next generation technologies started to come to fruition. The most important is probably a method known as Solexa, which forms the basis of the technology from sequencing giant Illumina.
In the mid-1990s, Cambridge scientists Shankar Balasubramanian and David Klenerman were working on a way to study the motion of DNA polymerase as it added a single ‘letter’ onto a growing strand of DNA stuck to a surface. Chatting in the pub, and inspired by Cambridge’s rich history of innovation in DNA research, they realised that the technique they were using for their rather niche molecular biology research could be turned into a completely new technique for DNA sequencing.
Sequencing by synthesis technology, or SBS, works by breaking the DNA you want to read into short fragments and adding special adapters onto the ends of each piece. These fragments are then placed in a special glass slide, coated with little stretches of DNA known as oligos, which pair up with the adapter sequences and capture the DNA fragments on the slide. Next, theres a clever process that makes millions of identical copies of each DNA fragment, all stuck in close proximity on the slide.
After that comes the actual sequencing, where DNA polymerases make a copy of each of these millions of fragments, adding fluorescently labelled bases one at a time, according to the sequence of that fragment. Each of the four bases is labelled with a different colour, emitting a flash of light as it’s added that can be detected with a highly sensitive camera.
By watching the patterns of flashes, it’s then possible to figure out the DNA sequence of each cluster of identical fragments, and then use computer analysis to piece them all together to get the overall full length sequence you started with. If you want to know more about how this works, there’s a video on the page for this podcast at Geneticsunzipped.com
The first Solexa Genome Analyzer machines came on the market in 2006, and although they were initially only capable of generating pretty short reads, they were improving fast. Illumina snapped up the company in 2007, quickly growing to become the dominant player in the space and currently generating more than 90% of the world's sequencing data.
There are now several types of next-generation sequencing technologies commercially available with differing technical details but similar overall processes that allow thousands or even millions of DNA molecules to be sequenced simultaneously in real-time using parallel reactions.
Next generation sequencing lit a rocket under the field of genomics, slashing the cost and time taken to sequence individual genomes from humans or any other species from months and millions down to hours and a few hundred dollars. Next generation sequencing technology has truly unlocked the power of genomics and transformed the life sciences.
So what’s coming next?
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