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Approaching Sanger Range, 454 Plans to Extend FLX Reads to 500 Base Pairs in '08

454 Life Sciences is planning to update its recently launched Genome Sequencer FLX by the end of next year, bringing the system’s read length into the realm of Sanger sequencing, In Sequence has learned.
“It’s not [going to be] a new instrument,” Mary Schramke, 454’s vice president of marketing, told In Sequence last week. “It’s the platform that people have bought, it’s the one that they can stick with, and we will be making improvements constantly.”
Specifically, the planned update will enable the FLX to generate 500 base-pair reads, one million reads per run, and have an output of 500 megabases of sequencing data.
At present, the instrument, which 454’s marketing partner Roche launched earlier this month, delivers read lengths between 200 and 300 bases, more than 400,000 reads per run, and yields 100 megabases of data per run.
Stephen Kingsmore, president of the National Center for Genome Resources in Santa Fe, NM, presented the planned instrument specifications as part of a comparison between different next-generation sequencing platforms in a talk he gave at this month’s Plant and Animal Genome conference in San Diego. He said he obtained his information directly from 454, and Schramke confirmed with In Sequence last week that “these are all targets that we are working to achieve by the end of ’08.”  She stressed that this deadline may change but called it “not unreasonable.”
454 has already presented pilot testing data from the FLX at conferences. At the Genomes, Medicine, and the Environment conference last fall, for example, the company showed that the instrument can generate 500 base-pair reads using 200 flow cycles.
“We obviously have to make that robust to where it generates the very high-quality reads at the 0.5 percent error rate that we are currently achieving with the GS FLX,” Schramke said.
Schramke did not elaborate on how exactly 454 is planning to achieve this, other than saying that it will be a combination of improvements to the software, reagents, and consumables.
Earlier this month, Bruce Roe, director of the Advanced Center for Genome Technology at the University of Oklahoma and a 454 user, told In Sequence that his center already routinely obtains 300 base-pair reads, and up to 500 base-pair reads, on the GS 20, the predecessor of the GS FLX.

“Until 454 can solve the technical problems with the [homo]polynucleotides, … Sanger [sequencing] will stay in business,” he said.

The bottleneck for handling longer reads, he said, is the assembly software. “As you get reads further out, you get slightly less signal, and then it becomes difficult to interpret, and so it becomes a software problem to figure out how to interpret the reads, or the flows, and how to weight them in the assembly,” he said in the interview.
His center is currently testing new software for 454. “They are doing a very good job of trying to improve the software for these longer reads,” Roe said.
With reads of 500 base pairs, 454’s updated instrument will be approaching the 800 base-pair reads of Sanger sequencing. Those longer reads would make the machine more useful for de novo sequencing.
“An improvement to 500 base pairs would be excellent, since assembly would be much, much easier,” said Axel Strittmatter, manager of the Göttingen Genomics Laboratory at the University of Göttingen in Germany, and a recent 454 customer, in an e-mail message. However, he remains doubtful that the FLX would replace Sanger sequencing even with the longer read length because of its slightly higher error rate when reading stretches of homopolymers.
“Until 454 can solve the technical problems with the [homo]polynucleotides, …Sanger [sequencing] will stay in business,” he said.

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