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12th October 2009
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The $1000 genome in sight with the latest technology

Michael Gross, 12 October 2009

Decoding the first complete human genome, that of sequencing pioneer Craig Venter, is estimated to have cost around $10m. The second, that of James Watson, Nobel laureate and co-discoverer of DNA’s structure, cost around a tenth of that, and by June 2009, current market leader Illumina offered a full genome sequencing service for just $48,000. Meanwhile, pundits, like the genome pioneer George Church of Harvard University, US, predict that we will soon see the price drop to $1000/genome, and that at this point, genome sequencing will be as revolutionary as the introduction of affordable desktop PCs. Complete Genomics, where Church is a member of the scientific advisory board, recently announced that it had sequenced 14 human genomes this year for as little as $5000/genome.

The sequencing of all 3bn base pairs of the human genome should not to be confused with services scanning the genome for common variants – single nucleotide polymorphisms (SNPs) – which are already available for less than $100 from companies like 23andme and Knome. Initially hyped as a panacea for pharmacogenomics, this type of analysis has revealed very little information of medical interest. Experts now agree that complete genomes, and lots of them, will be needed to understand why individuals are susceptible to certain diseases and respond differently to some drugs.

Single strands

But why is the cost and time required to sequence a complete genome falling so fast and dramatically? The current ‘second generation’ of sequencing machines typically use DNA synthesis with fluorescent bases, so the machine ‘watches’ as new building blocks are added to a growing strand and notes the colour of each added unit. Unlike the living cell, which only needs one double helix to read out the information stored in it, sequencing still relies on a multitude of identical DNA strands.

So the first big improvement sequencers need is to mimic nature and read the sequence from a single molecule. Several companies are now developing single molecule sequencing methods. The first one to reach the market is Helicos’s HeliScope system, which is based on sequencing technology invented by Stanford University, US, engineer Stephen Quake. Helicos vp and chief scientific officer, Patrice Milos, says: ‘Single molecule sequencing will indeed deliver. Over the next two to four years, we at Helicos indeed see a path to a complete genome sequence, at 20 times coverage using the HeliScope, that will be well below the $1000 threshold.’

In August, Quake became the eighth person to have his genome published (Nature Biotechnology 2009, 27, 847), an announcement that garnered much media attention as only three names appeared on the paper. While this is the first complete human genome sequenced by single molecule technology, commentators around the world quickly jumped on claims of reduced cost.

At just under $50,000, Quake’s sequence is no cheaper than the commercial offer available from Illumina, using established technology including amplification. Genomics blogger Daniel MacArthur commented: ‘It appears that formal entry into the human genome sequencing race merely requires generating a genome sequence of the standard that second-generation sequencers were achieving in early 2008, at the same price that they’re charging right now. That’s a fairly uninspiring goal.’

Meanwhile, other biotech companies are also developing single molecule technologies. Pacific Biosciences (PacBio) based at Menlo Park, California, is developing what they call SMRT (single molecule real time) DNA sequencing, a technique based on DNA synthesis taking place in a tiny hole.

Two novel tricks enable SMRT to sequence a single strand of DNA. As the strand grows, the nucleotides’ fluorescent labels, which are attached to the phosphate groups, are cleaved off. This reduces background ‘noise’ to produce a cleaner signal, so that fluorescent markers from a single DNA strand can be successfully analysed. And to ensure that only one strand is growing at a time, the well is so small that only a few strands of DNA can enter. Diffraction effects within the tiny hole increase SMRT’s sensitivity by making it unlikely that more that one fluorescent building block will be present at any one time. Unlike Helicos’s technology, which has been criticised for extremely short reads, typically around 32 bases, SMRT sequencing is designed to produce long reads and good processivity.

PacBio’s ceo Hugh Martin says: ‘When we talk to customers, what they are excited about with the next generation of DNA sequencing technology is… the availability of very long, high quality reads delivered in a very short amount of time. They are completely agnostic as to how you get those properties.’

Following the splash caused by Quake’s genome, PacBio responded swiftly, announcing that it had acquired $68m in additional funding, raising total funding to $188m. New investors include the Wellcome Trust and Monsanto. The company plans to release a commercial version of its single molecule sequencer in the second half of 2010.

John Milton, who played a key role in the development of the Solexa technique which was bought up by Illumina and is currently leading the market, is now vp of Research at Oxford Nanopore Technologies (ONT), a spin-out based on the nanopore research of Hagan Bayley at Oxford University, UK. He is leading the team developing a new single molecule sequencing technology and believes that they must go one step further than Helicos and PacBio.

‘The current [second] generation of sequencers requires amplification and light detection,’ Milton explains. ‘Then the next step is to use single molecules and get rid of the [polymerase chain reaction] PCR. But the ultimate goal must be to remove the light as well and to develop a technology that analyses single molecules without optics.’

Using electronic detection instead of optical devices, Milton says, the sequencers can be simplified much further, and could become truly affordable to medical practitioners and research labs alike.

ONT’s idea is to have a modified version of a natural membrane pore sitting in a membrane with an electric potential driving ions through the channel. Attached to the entrance to the pore sits an exonuclease, an enzyme that chops bases off the end of a DNA strand one-by-one. The electric field pulls the nucleotide released by the enzyme through the pore and when it passes the narrowest point – an artificial bottleneck created by incorporation of a cyclodextrin derivative – it blocks the current passing through the pore, creating a characteristic dip in the recorded current trace. Using this technique ONT researchers can not only distinguish between the standard four nucleotides, but can also identify methylated bases, which is important for an improved understanding of epigenetics – the regulation of gene activity. ‘We’re on the cusp of a breakthrough here, which allows digital electronic readout of sequences, and whose performance will start where improvements to current technology level off,’ says ONT’s ceo Gordon Sanghera.

While the Helicos sequencing system is still the size of a large fridge and comes with an inbuilt granite slab to stabilise the laser microscopes, ONT is confident that its technology, which will be marketed by current leader Illumina, will ultimately fit into a small desktop box that will be user-friendly and could become a standard tool in many hospitals. For instance, the company points out that oncologists have recently been sequencing patient’s tumour genomes and comparing them with healthy cells in order to spot the tumour’s Achilles’ heel.

So how long before general practitioners are sequencing a patient’s genome in their surgery or sequencers are found in hobby labs? ONT does not want to reveal time plans or prices yet, but it quotes Illumina president, Jay Flatley, who said earlier this year that babies born from 2019 onwards will routinely have their genomes sequenced at birth. ‘We will reach a tipping point as with PCs and the web,’ Sanghera says.

Michael Gross is a science writer based in Oxford, UK.

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