I recently came across an interesting article in the July/August 2010 issue of Australian Life Scientist. It discusses technological advances in the field of genome sequencing that are overcoming cost and speed limitations and opening the door to new applications. Here is an overview of what was discussed:
It took nearly two decades to go from the release of the first semi-automated genome sequencer in the mid-1980s to the launch of Roche’s flagship 454 FLX next generation sequencer in 2005. The 454 is now one of three major players in the next gen market whose impact on the world of genomics cannot be underestimated. Just five years later we are poised to embrace another new wave of sequencing technology. Next gen sequencers, exemplified by Illumina's Solexa Genome Analyzer and the APplied Biosystems SOLiD System, together with 454, are likely to continue to be adapted for myriad use rather than being superceded by the next next gen technology platforms.
The new wave of sequencers, sometimes called the third gen, are creating deal of excitement because they will likely enable scientists to reach the goal of the $US1000 human genome. The third generation of sequencing technology sees single molecules of DNA being sequenced without the need for cloning or PCR amplification and the inherent biases these procedures introduce. There are generally two types of detection methods for single molecule sequencing: those that rely on fluorescence and CCD capture, and those that don’t. Instruments that use the first of these detection methods include the Helicos Heliscope, launched in 2008; Pacific Biosciences single molecule real time sequencing (SMRT) machines, which have been shipped to their first customers; and Life Technologies-VisiGen system, which relies on fluorescence resonance energy transfer (FRET), and Life Technologies expects the first instrument will be placed later this year.
The third generation
The two non-fluorescing technologies operate via quite different methods, with detection systems based on tiny changes in electrical current or pH, thus removing the most expensive components, and associated costs, of sequencing instruments. Both nanopore sequencing, from Oxford Nanopore Technologies (ONT), and Ion Torrent, from Mass Genomics (which was just acquired by Life Technologies), are based on silicon chips. The ONT chip contains hundreds of wells each covered by a lipid bilayer that contains a nanopore – which is a hole around one nanometre in diameter – with each pore an individual electrical channel.
Sequencing is based on exonuclease cleavage of the single DNA strand and detection occurs when the cleaved nucleotide falls through the pore, transiently disrupting the current. The change in current amplitude is unique for each base (A,G,C,T and 5-Methylcytosine - the direct reading of which is unique to nanopore technology). ONT plan to commercialise their nanopore sequencing system by the end of 2010.
Ion Torrent, while also relying on advances in semi-conductor technology, sequences by monitoring DNA synthesis. Single types of nucleotides are sequentially flooded across the chip. Nucleotide incorporation into the new DNA strand results in the release of a H+ ion, which is detected by the pH sensitive dielectric layer. These breadbox–sized benchtop instruments come with an iPod, pre-installed with an application to monitor runs and cycles in real time. The Ion Torrent Personal Genome Machine (PGM) sequencer is just making its way into American research labs now.
Second generation sequencing redux
The third generation upstarts may be on their way, but that doesn’t mean second generation sequencers don’t have more yet to give. Improvements to the technology continue to be made on a number of fronts. They include an increase in the number of wells/reads per plate, superior base-calling algorithms and CCD detection rates and resolution (so the depth of sequencing required can be reduced for the same accuracy), and creation of scaled-down versions of instruments that, cost-wise, will put them in reach of the smaller research laboratories.