Move over Skippy - big-time sequencing has arrived

"Since we can't really do much about Alzheimer's, I didn't want to know whether I was at risk," says Professor James Watson, Nobel laureate, co-discoverer of the double helix and proud owner of his own personal genome on DVD.

According to reports, there could be up to 50-odd dodgy bits hidden away in his genome, but being the pragmatist that he is, he's not too keen on investigating them at his time of life.

The hugely complex and intricate job that it is to sequence a human genome is not likely to be repeated yet in Australia, but we are moving ahead. The Australian Genome Research Facility (AGRF), in association with the Baylor College of Medicine in Houston, has just finished sequencing the genome of the Tammar wallaby, to date our only attempt at genetic sequencing on this scale.

In April this year, the AGRF purchased a Roche Diagnostics Genome Sequencer FLX system, a major piece of equipment that looks set to make what was formerly impractical practical.

Kirby Siemering, the AGRF's business development manager, says the equipment now moves projects that were in the past enormously difficult, mainly due to funding and time constraints, into the realm of possibility.

The technology certainly has limitations, but the facility is already working on resequencing projects that should allow Australian researchers to join the rest of the first world in undertaking work of this nature.

These include resequencing the yeast genome with the Australian Wine Research Institute; resequencing some E.coli genomes with the University of Queensland's School of Molecular and Microbial Sciences to look for differences between pathogenic and non-pathogenic strains; and fascinating work with UQ's Professor John Mattick, looking for rare changes in the sequences of genes to understand the role of RNA editing in the brain.

"What this sequencing technology allows you to do is sequence individual molecules, so if you put the products of a PCR reaction into the sequencer you can get up to 400,000 individual sequence reads from one run," Siemering says.

"What that allows you to do is find one variant amongst 400,000 individual molecules. It's very powerful." Siemering says. "Cancer researchers could be looking for the early appearance of rare somatic mutations in a tumour, for instance.

"So if you are interested in a particular gene or exon, you can take a whole lot of cells from the tumour or the brain or whatever it might be, PCR the exons and then you have millions of fragments that can be individually sequenced."

The AGRF is working with researchers on such diverse areas as sequencing small genomic fragments from the elephant and on transcriptomics with the Australian Centre for Plant Functional Genomics.

"It's a very flexible instrument in that, in addition to genome sequencing, it can do a lot of applications that you can currently do on microarrays, so people are very interested in using it for gene expression studies, for example," he says.

"With this technology you can do digital gene expression, where you essentially use RNA, which is converted to cDNA, put that into the instrument, get 400,000 reads and then you just literally count the sequences.

"And because it's a sequence you can tell what gene it came from by mapping it back to a genome. If in one sample you have 10 sequences from gene A and in another you have 1000 sequences from gene A, you can say that gene's been a hundred-fold upregulated.

"With microarrays you have to read fluorescence, which is an indirect measure, and then do all the normalisation. With this sequencing technology, you are literally counting molecules of RNA that were in the starting sample."

Speed and cost

One of the great benefits of the new technology is speed, and with that, cost effectiveness. Siemering says the instrument currently allows researchers to do around 100 million bases of sequencing in one run.

"It allows you to do in less than a week the equivalent of what it would take a centre like ours months to do with the normal Sanger technology that we've used in the past," he says.

"And it's getting to an order of magnitude cheaper to do, when everything's taken into account. It's quite a disruptive technology that is making things that were impractical, practical."

Sequencing is just one of the technologies the AGRF is exploring, however. Its other main areas of expertise are genotyping and microarray analysis. It is also moving into the areas of epigenomics and structural genomics, Siemering says.

"We are working with various researchers on whole genome methylation, using tiling arrays," he says. "It involves ChIP-chip technology: using an antibody to target the methylated DNA. You pull down the methylated DNA, label it up and put it onto a whole genome tiling array and look at what bits light up.

"We are also interested in a related area, structural genomics, using array CGH (comparative genomic hybridisation) to look at chromosomal variations: translocations, insertions, deletions.

"With these extremely dense microarrays you can do that at extremely high-resolution compared to good old FISH (fluorescent in situ hybridisation) and cytogenetics. They are some of the newer areas we are pushing into."

The AGRF is also doing some more commercially oriented work, especially in biotech and pharma. Microbiology is also opening up for the AGRF, which has recently entered a partnership with a new company called Microgenetix, aimed at nucleic acid-based microbial identification.

"The microbiology industry has been working with pretty much the same technology for decades, phenotypic technology, which focuses on what a microbe looks like and what biochemical reactions it has. You do these batteries of tests and at the end you are only right around 70 per cent of the time. It's not a terribly flash technology.

"What we are doing is using gene sequencing to identify microbial species based on their gene sequence, which is a very accurate way of doing it. It's a methodology which is currently redefining how taxonomy is done in the microbial world.

"Now we are offering that service through our partner company - doing all the gene sequencing here. We are starting off with species identification and are now developing technologies that will allow strain typing for epidemiological investigations. It's a very exciting time in genomics and genetics."