What is DNA sequencing?
DNA sequencing involves reading the order of nucleotides (A, C, T and G) of a DNA strand. This may be 5 nucleotides long, or it might be 5 million nucleotides long!
All living organisms have DNA (or similar molecules) – which contain the ‘instruction book’ for how that organism lives and replicates.
Just like we can read the DNA sequence of humans, we can use the same technology to read the DNA of bacteria and viruses. Our research group specialises in reading the DNA of bacteria.
The Sanger method, developed by Fred Sanger at the University of Cambridge, was the first technique that allowed us to read the sequence of DNA on a large scale. It is extremely accurate, and for many years it was the only method available. We still use it to accurately sequence smaller strands of DNA (up to a few thousand nucleotides). However for an entire genome, of millions of nucleotides, it could take months to years to analyse the DNA sequence in this way, so for this, newer technologies are used.
For A-level students, Sanger sequencing is the method that you may be taught about.
DNA sequencing using Next-Generation Illumina/Solexa Technology
The Illumina MiSeq and HiSeq machines are the most commonly used large-scale DNA sequencing machines at the moment. Our group has conducted most of it’s research in the last 10 years using this technology.
Briefly, this technique involves cutting very long DNA strands into very small fragments, reading the sequence of all of these strands separately, and then doing a ‘jigsaw puzzle’ to piece them all together again. In slightly more detail – small fragments are attached to a glass plate, called a ‘flow cell’. Imagine a very sparse ‘seaweed forest’ of single strands of DNA, stuck to a glass bottom. Millions of different DNA strands are stuck on, and then copied, so that there are now different ‘clusters’ of identical DNA strands, together in a very dense forest. Fluorescent chemicals are added which stick to the DNA, and flash when a light is shone at them. The Illumina machine takes thousands of ‘flash photography’ images, and the information in the photographs can be used to piece together the information about the original DNA sequences.
It might seem a bit odd to read lots of tiny pieces of DNA and try and put them together, rather than just reading one long piece. But it’s really hard to sequence long pieces of DNA, so this was the only method we could use to sequence DNA fast enough for many years. It’s a bit like trying to read a massive book – read by one person, it may take a few days. If you cut it up and ask 5 million people to read a small fragment of 200 letters, you can get the information in seconds.
It’s a very hard process to explain! In fact, even many of our researchers using the technology will struggle to understand it in detail…
Here is one video explaining the process (in this case they are using it to analyse human DNA).
Here is the video from Illumina, explaining the technology.
(This is a video that requires in-depth molecular biology knowledge to understand).
If you’ve seen the Sequence VR experience – this is a small explanation of how the digital images are put together to read the DNA strands (again this isn’t straightforward!)
You can read a bit of the story about how the Illumina/Solexa technology was invented at Cambridge University here:
DNA sequencing using Pacific Biosciences (PacBio) Technology
PacBio sequencing can read very long strands of DNA very accurately. Our research group has used it to read the sequences of small loops of bacterial DNA , called plasmids, and understand how these can make bacteria resistant to antibiotics. Whilst it is extremely accurate, the machines required to perform the sequencing are large, and it is quite slow and expensive to read large numbers of DNA sequences. This is why we still use other technologies in day-to-day work and large-scale projects. We do use PacBio for specialised projects.
DNA sequencing using the Oxford Nanopore Technology
The MinION is a very new, pocket-sized DNA sequencer, developed by Oxford Nanopore in the last few years. It is designed to read the sequence of very long fragments of DNA quickly and cheaply, and is a very exciting new development. One main benefit is that it is very portable, so it can be taken abroad, and out in remote environments. We’ve been using the Nanopore technology to speed up DNA analysis, aiming to be able to get the results of bacterial DNA tests on the same day as a sample is taken. At the moment, we use other technologies to do large-scale sequencing (when we want to run hundreds of samples at once). But as a recent development, the Nanopore technology is improving all the time, and we hope to be using it a lot more in the future.
Some work done from our research group using Oxford Nanopore Technology
How DNA sequencing using Oxford Nanopore technology works:
We will be demonstrating how DNA is sequenced using the Oxford Nanopore MinION at the Royal Society Summer event.
You can read more about how the Nanopore was invented at the University of Oxford here:
Jonathan Eisen summarises a history of DNA sequencing, and recent developments