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Helicos Talks About Homopolymer Troubles; Solution Paves Way to 2007 Beta-Testing

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Helicos BioSciences last week for the first time publicly acknowledged that until last year it had been “unable to address homopolymer sequencing,” but said it has found a solution to this problem.
 
In a presentation at last week’s Advances in Genome Biology & Technology conference in Marco Island, Fla., Bill Efcavitch, Helicos’ senior vice president of product research and development, said the company has developed a proprietary nucleotide-polymerase pair where the nucleotides prevent the polymerase from adding more than one base per reaction cycle.
 
Although Efcavitch would not elaborate, the novel nucleotide analogs could be reversible terminators, similar in principle to the ones Solexa developed for terminating synthesis after each step. [Illumina acquired Solexa last month.]
 
Initially, he said, the company tried to control the kinetics of the polymerase reaction but “could not find reliable conditions” for stopping the polymerase after adding one nucleotide when it encountered a homopolymer stretch.
 
Last fall, In Sequence’s sister publication GenomeWeb Daily News reported that Helicos was working with Floyd Romesberg, a professor of chemistry at the Scripps Research Institute, to develop an improved DNA polymerase that can incorporate modified nucleotides (see 3/10/2006).
 
According to Efcavitch, Helicos, has so far shown it can sequence a run of five identical bases accurately and plans to extend this number to 10.
 
He also described the company’s platform, called the HeliScope, and its expected performance.
 
He said Helicos has been developing several sample-preparation methods, the simplest of which is a six-hour protocol during which DNA is sheared and cut to fragments of 100 to 200 nucleotides in length, to which an enzyme adds a poly-A tail. These DNA templates are then added to a poly-T-primed surface, although Efcavitch said the company might develop other surfaces for different applications.
 
The density of the template is going to be one per square micrometer, or 100 million templates per square centimeter, he said. Generation one of the HeliScope will have a total of 3 billion templates on two separate flow cells.
 
Subsequently, the DNA is sequenced using fluorescently labeled dNTPs. Since the company only uses one fluorophore for all four bases, it has to run four reaction cycles in order to read one base.
 
After each cycle, which needs less than 20 minutes, the instrument takes 69,000 images, lasting 45 minutes. As a result, determining a single base requires more than 4 hours.
 

Helicos plans to place beta-instruments with early-access customers by the end of the year.

Helicos plans to run two flow cells in parallel, such that chemical reactions can take place on one while the other one is imaged, similar to ABI’s approach for running its SOLiD platform.
 
At the moment, Helicos reaches an average read length of 29 bases, with some reads going up to 60. However, Efcavitch did not mention the error rate of the reads. The company, he said, is working on improving the efficiency of the chemistry and increasing the number of cycles.
 
Helicos plans to place beta-instruments with early-access customers by the end of the year, Efcavitch told In Sequence.
 
Last month, Helicos also received a patent for a method that solves “the problem of determining the number of nucleotides in a homopolymer stretch” (see patents).
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