April 14, 2006 | “To some extent, it’s like 1990 all over again,” says Rick Wilson of the Washington University School of Medicine. Back then, investigators involved in the human genome project (HGP) were ramping up sequencing technology to churn out DNA sequence faster and cheaper.
And if the presentations at February’s Advances in Genome Biology and Technology (AGBT) are any indication, the race to generate fresh approaches to produce more sequence for less is far from over — and looks to be heating up. Some technologies, including those offered by 454 and Solexa, are ready to roll. Others are a bit further off, but all are making “incredible progress,” says Jeffery Schloss, program director for technology development at the National Human Genome Research Institute (NHGRI). “Everybody’s energized because they see the competition succeeding.”
“There’s been a push over the past several years to take sequencing from something of a boutique industry and drive it to really high throughputs because the need for sequence information is basically insatiable,” notes Elaine Mardis, another denizen of Washington University and co-chair of the meeting.
Fueling this recent technological explosion, in addition to data lust, is a healthy dollop of government funding. “NIH came up with $70 million, which got the creative juices flowing,” says George Church of Harvard Medical School. The first time around, he notes, the HGP “emphasized production and de-emphasized technology.” Now the NHGRI-sponsored programs are spurring the development of technologies designed to bring us the $100,000 genome in fairly short order and the $1,000 genome a decade hence (see Church Inquiry Gets Personal, March 2006 Bio-IT World, p. 6).
NHGRI is trying to drive the technologies to the point where they’ll help the community meet its sequencing needs. “If we give people grants and they don’t get any further than a publication in Analytical Chemistry...we’ve basically failed,” says Schloss. “We want these technologies out there.”
And the “needs” of the community are changing. “There’s a lot more to do than just sequence next platypus genome,” says Wilson. Many researchers are turning their attention to “resequencing” whole or partial genomes that have already been “completed” to search for SNPs and other alterations that correlate with disease.
Focus on Resequencing
Although Solexa’s mission is to provide machines that will produce 1 gigabyte of sequencing data for a few thousand dollars, Solexa chief scientist David Bentley described the organization’s success in resequencing a segment of human chromosome 6 — a fairly typical chunk of human DNA in terms of GC content and gene density. Using the current Solexa instrumentation (see Solexa Readies 1G Genetic Analyzer, Feb. 2006 Bio-IT World, p. 24), Bentley says his colleagues detected all known SNPs in the region and correctly read repeat sequences that can flummox other technologies.
While Solexa’s machines begin shipping this year, other investigators presented approaches that are a bit more far out. Michael Metzker of Baylor College of Medicine discussed progress toward cyclic reversible termination — a “sequencing by synthesis” technique that uses modified fluorescent nucleotides to block the addition of more than one base at a time, allowing researchers to make a nucleotide call before the reaction proceeds. The technique currently allows one to read about 50 bases, but Metzker’s group is aiming to read lengths of 300 bases or so.
Susan Hardin of VisiGen Biotechnologies presented a base-calling method that relies on FRET between a pair of fluorophores — one attached to the polymerase and a second attached to the incoming nucleotide. Although she predicts the maximum base read will be only about 50 nucleotides, Hardin envisions scaling up the technology to produce sequence data at a rate of 1 million bases per second.
And when it comes to resequencing, traditional capillary electrophoresis is not out of the game. Applied Biosystems has introduced TargetSeq, a new software system designed to optimize the ABI 3730 instruments owned by most sequencing centers and many independent investigators for sequencing individual exons or other small DNA fragments. By reducing the length reads from the 800 bases preferred for de novo sequencing to, say, the 400 needed for resequencing, researchers should be able to double their daily throughput, perhaps reaching 2.8 megabases per day.
De Novo Not Dead
Of course, de novo sequencing isn’t dead, and a forerunner in the field is 454 Life Sciences. Its machines (see Fantastic 454, Sep. 2005 Bio-IT World, p. 1) are already being deployed in several labs. “They’re very powerful for just blasting through bacterial genomes,” notes Wilson. “One run and the genome’s finished.” Indeed, Michael Egholm, 454’s VP of molecular biology, hopes the company will become “the only game in town for bacterial de novo sequencing.”
John Williams of LI-COR Biosciences described a method for sequencing single DNA molecules that could reach up to 20 kilobases in length. The system involves using an electric field to flow labeled nucleotides over an immobilized polymerase and template. Although the company is still a year away from performing any actual sequencing, Mardis notes that a technique that permitted long, continuous reads would be great for determining whether a cluster of SNPs comes from one parent.
Whether one of these technologies will emerge as the “winner” — for either de novo sequencing or resequencing — may ultimately depend on a balance between quality and cost. “At end of the day, we’re all publicly funded or charity funded,” says Chris Clee of the Wellcome Trust Sanger Institute. “We have to make every pound and dollar count.”
In that case, keeping more technologies in the mix might help. “If these companies are competing with each other for cost, that will be good for all of us,” says Barbara Wold of the California Institute of Technology.
And different platforms will lend themselves to different applications. “It’s not necessarily a race to be the provider of a sequencing platform,” says Mardis. Rather researchers will choose a technology based on its applicability of their projects. In the end, Mardis predicts, “we’re going to move away from the situation we’ve been in where we have one primary manufacturer of sequencing equipment and reagents.” Which suits researchers like Church just fine. “Right now it’s exciting because we have so many options,” he says. “That makes some people nervous because they don’t know what the best system might be. But to me it’s like being a kid in a candy shop. I hope it stays this way forever.”
Sidebar: Toward the $1,000 Genome
A pair of sessions at the AGBT meeting titled “Toward the $1,000 Genome” were devoted to next-generation technologies But what exactly is a “$1,000 genome”? The term suggests different things to different people. “To me it means that you can sequence echidna for 1,000 bucks,” says Rick Wilson, who’s championing a proposal to nail the genomes of unlikely creatures such as the platypus and its egg-laying cousin the echidna.
It could also refer to an ability to sequence a human genome with enough coverage, he says, “so you can map back to the reference sequence and say, ‘Here’s a deletion, here’s an insertion, maybe this is why this person has this phenotype.’”
Technically it refers to both, says Jeff Schloss, who oversees the relevant NHGRI program. The term “$1,000 genome” is shorthand for reducing the cost of sequencing a mammalian-sized genome — now around $10 million — by 10,000 fold. But for grantees who are more interested in more targeted, medical sequencing efforts, the same goal applies: lowering the cost of resequencing by four orders of magnitude. The jazzier terminology has stuck, says Schloss, because “it’s a nice, round, easy-to-understand target, and it happens to be about the cost of a standard medical test.”
Researchers at the conference seemed optimistic about reaching that goal. “From what I heard [during the meeting],” says George Church, “I see nothing that indicates to me that we shouldn’t be able to do a $1,000 genome at high coverage, with high accuracy and close to zero errors.”
What’s more, he sees no reason to stop there. “We should not rule out the possibility of an amazing genomic test for less than $100,” says Church. Such a tool might allow doctors to screen for cancer at key genomic positions. If the price were right, says Church, “I wouldn’t mind having my blood regularly sequenced for prostate cancer.”
And by the time the community hits the target, who knows what $1,000 will buy? “It’s attractive to think that 10 years from now, high-school students will be doing science-fair projects that begin with going out and determining the sequence of something,” says CalTech’s Barbara Wold. “Let’s face it: $1,000 isn’t what it used to be.” -- K.D.