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Sequencing's new race
Erin Podolak
BioTechniques, Vol. 48, No. 2, February 2010, pp. 105–111
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Next-generation sequencing has pushed forward the boundaries of genetic research and enabled the completion of a rapidly growing number of whole-genome sequencing projects. But the impending arrival of third-generation DNA sequencing technology could change the landscape yet again.

In the late 1990s, scientists J. Craig Venter and Francis Collins became household names during the epic race to sequence the human genome. Eventually that race ended in a tie in 2000, but similar levels of fame and publicity—not to mention millions in potential revenue—await the company or individual that wins sequencing's latest race: to develop and implement a so-called third-generation sequencing system.

The sequencing community turned its sights toward the promise of third-generation sequencing instruments less than five years ago, when a number of companies started promising single-molecule sequencing instruments that could provide faster, cheaper genome sequencing—instruments that would finally enable the burgeoning field of personalized medicine and take new fields of study such as transcriptomics to the next level.

So strong is the interest in new sequencing technologies that the National Institutes of Health (NIH) is supporting the race toward third-generation technologies through their $1000 Genome Initiative, which provides funding to companies and individuals developing innovative solutions aimed at rapid, efficient DNA sequencing. Given the frenzied pace of development and the millions of dollars in financial support, many suspect that it is only a matter of time before someone crosses the finish line, commercializing a third-generation sequencing system that will finally give researchers a full human genome sequence for less than $1000.

Slow, long reads: the starting line

Frederick Sanger was awarded part of the 1980 Nobel Prize in Chemistry for his development of a method to sequence DNA. His approach uses gel or capillary electrophoresis to separate DNA fragments of differing lengths that have labeled dideoxynucleotides incorporated at the ends. By identifying each labeled nucleotide in the resulting DNA ladder, the ordered sequence of any DNA fragment can be determined. After nearly 30 years, Sanger sequencing, as it has come to be known, is still being used by many researchers for its simplicity and effectiveness along with its ability to accurately determine the sequence of long stretches of DNA.

A few years after Sanger won his Nobel Prize, developers at Applied Biosystems launched an automated DNA sequencer based on his method, which used fluorescently labeled dideoxynucleotides. Sanger's sequencing-by-synthesis approach to DNA sequencing, along with the newly developed automated instruments, would serve as the enabling technology for the Human Genome Project. Although the method has proved durable, reliable, and accurate, it suffers from being slow, expensive, and relatively low-throughput—key reasons why Collins' government-funded researchers required $3 billion over 13 years to generate their draft map of the human genome.

Recent advances in the Sanger methodology and associated computational assembly tools, along with the availability of the draft human genome sequence, enabled the assembly of another human genome sequence in 2007, using the dideoxy approach. This time, though, the genome was of a single individual: J. Craig Venter (1). Despite the obvious success of the Human Genome Project and the other recent whole-genome sequencing efforts, Sanger sequencing is slowly becoming outdated. Costs remain high and throughput is not high enough to support the growing interest in personalized medicine and the desire to uncover the genetic basis of human disease. Since these efforts could require tens of thousands of individual genome sequences, geneticists have been searching for alternatives.

Claire Wade, a professor of veterinary science at the University of Sydney in Australia, recently completed the sequencing of the horse genome using Sanger methodology (2). “The horse was one of the last sequencing projects to be fully conducted using Sanger sequencing,” says Wade. “It represents the peak of our understanding of mammalian assembly on that platform.”

The next generation: ready to go

As it turns out, the desire to improve upon the Sanger approach and related instrumentation over the last decade has led to the development of a new generation of sequencing platforms that are currently used in labs around the world. But even these newer and faster ‘next-generation’ platforms might merely be holding scientists over until the arrival of third-generation technologies.

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