The next generation in sequencing is SOLiD

Applied Biosystems is set to acquire Massachusetts-based genetic analysis company Agencourt Personal Genomics (APG) and hopes to bring the company's novel next-generation sequencing technology to market next year.

Visiting Australia recently to introduce the local sector to the potential of the new technology, Applied Biosystems' (AB) senior director for market development and strategic alliances, Andy Watson, said the technology was capable of producing a great deal more data than anything else on the market while dramatically reducing the cost.

Impressively named the Supported Oligo Ligation Detection (SOLiD) process for massively parallel sequencing by stepwise ligation, the technology is an extremely high throughput approach to DNA/RNA analysis, Watson said. It is expected to be applicable to many genetic analysis applications, including de novo genome sequencing, medical sequencing, high throughput gene expression and high throughput genotyping.

"These systems will have the capability of producing two orders of magnitude more data than any other instrument on the market," Watson said. "That changes the way the science can be done. It lets new applications become feasible and it simplifies a lot of the hands-on work."

Watson said some aspects of the technology were novel while others were improvements on regular fluorescence detectors with microscopes. "One of the novel aspects is the chemistry, the way the sequence gets read out," he said.

"Other chemistries up until now have been based on extending the DNA base by base and reading them out. This chemistry allows every fifth base of the DNA to be read out and this is quite a novel approach."

The APG technology combines single tube micro-bead sample preparation with high throughput multi-colour fluorescence imaging. The sequencing uses ligation probes, an approach that the company said would provide very high quality data compared to competitive polymerase-based approaches.

How the process works

Following creation of a library of DNA fragments, the second step in the process is to perform the familiar polymerase chain reaction (PCR) to amplify the fragments. The DNA is emulsified with micro-beads and then enriched.

The enriched beads are arrayed and then the iterative cycles begin. A sequencing primer is added and then a pool of fluorescently labelled oligo probes. The probes are eight bases long, with the fifth the interrogating base. The probes are made in sets of four, identifying with the nucleotides, at the fifth base from the 3' end. Each has a different colour dye.

The next step is to add a ligase - as opposed to the polymerase used in Sanger sequencing - as it is an oligo probe added to the primer.

The slide is then washed and imaged and the probe is cleaved. The repetition of these cycles is where the novel aspect of the technology comes in. When the fluorescent dye is removed, this leaves a native DNA strand, so that the ligate, read, and cleave cycles can be repeated.

This allows a signal to be generated at the 10th base in the second cycle, the 15th base in the third cycle, the 20th in the fourth and so on. The intervening bases can be determined using different nested sequencing primers.

The whole process is repeated until all of the intervening positions in the sequence are imaged. APG's process allows readings of millions of fragments of DNA, unlike the Sanger method which reads just one. This is the "massively parallel" part.

"The current electrophoresis technology will give you 96 samples in about 30 minutes, whereas this technology will give you a read-out of about a hundred million individual data points in the space of a few days," Watson said.

CE not forgotten

While Applied Biosystems is excited about the potential of the new process, champion stayers like capillary electrophoresis (CE) aren't exactly going to go away, AB's senior manager for genetic analysis systems, Carla Wike, said.

AB commercialised its first sequencer in 1984 and the technology is still going strong.

"Capillary electrophoresis will be around for many years and we'll continue to add more applications," she said. "Our strategy is to simplify the workflow and expand the range of applications."

Wike is currently working on developing an integrated sample preparation system, or blackbox, for the company's popular CE systems and hopes to include up to five steps in the sample prep process within one box.

Although the blackbox is still in its conceptual phase, the aim is to automate and miniaturise upfront sample preparation for CE. "It's not going to be an expensive piece of equipment and we expect to target labs that need automation but want to continue using existing CE equipment," she said.

New products on the market include a re-sequencing system called TargetSeq for AB's well-known 3730 target sequencer. The TargetSeq consists of a new software module to optimise the 3730 and 3730xl DNA analysers for high throughput re-sequencing or medical research sequencing of small exons and short DNA fragments. The system can produce 400 QV20 bases in 20 minutes or less.