By Branwen Morgan
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 the next generation of sequencing technology.
Professor Craig Cary, Director of the Sequencing Unit at Waikato University in New Zealand, says every time you move to a new platform you have to learn how to use it and that takes time. “There are punctuated steps in the evolution of technology. We are moving towards a whole new level of sequencing technologies where there are multiple affordable platforms; but all have inherent strengths and weaknesses.”
This means these 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Biosystems SOLiD System, together with 454, are likely to continue to be adapted for myriad use rather than being superceded by the next next gen. Indeed, despite the rapid changes in the sequencing landscape, Cary believes even the first generation capillary-based Sanger sequencing will persist. “No technology will suit all questions,” he says. “So I don’t see capillary reads ever going away, because they are single reads where each well is distinguishable and, for de novo sequencing, it’s the fidelity of Sanger sequencing that sets the bar.”
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. But to do this, the cost of the sequencing chemistry needs to drop to around $US0.0000005/base, or two millionths of a dollar per base (at a 10x coverage). Currently, the cheapest next gen sequencers cost about $US0.000001/base. And while this is a gigantic improvement on 1985 prices when sequencing cost roughly $US10/base, we are still well short of the price target.
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.
“There’s a lot of buzz around the Pac Bio platform, with reads expected to average over 1000 bases long; reaching and exceeding the read length of Sanger sequencing,” says Cary who travels to great depths, figuratively and literally – he has been to the ocean floor – and as far afield as Antarctica, to discover new forms of microbial life.
However, like the second gen machines, these systems require expensive excitation lasers, fluorescent reagents and CCD cameras. Another, not insignificant, cost comes from the storage of the digital images created by extremely high-resolution CCDs that equate to terabytes of data, not to mention the bioinformatics systems required to process the vast amounts of data generated.