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Helicos Demonstrates BAC Sequencing with 'Virtual Terminator' Nucleotides

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By Julia Karow

Researchers from Helicos BioSciences this week published a demonstration of the company's 'virtual terminator' nucleotides, which enable its single-molecule sequencing technology to read through homopolymer regions one base at a time.

In the paper, published online in Nature Methods this week, the scientists showed that they could use the reversible terminator nucleotides to sequence a canine bacterial artificial chromosome with even coverage.

According to the article, the four nucleotide analogs — one for each base — possess a free 3' hydroxyl group, a base modified with a propargylamine that is connected to a cleavable linker, and a fluorescent dye bound to an inhibitor that is attached to the same linker. Helicos calls them virtual terminators because they "block incorporation of a second nucleotide on a homopolymer template, despite possessing a free 3' hydroxyl," according to the paper. The blockage is released when the inhibitor and dye are removed by cleaving the linker.

The researchers tested their nucleotides by sequencing a previously analyzed 194-kilobase canine BAC, performing two-pass sequencing, where each molecule is sequenced twice, with a "low-capacity prototype sequencing instrument."

The data yielded a 15-fold median coverage of the BAC, which was even for "nearly the entire sequence" if non-uniquely aligned sequences were excluded. About 0.2 percent of the BAC was uncovered, the longest region being a 103-base pair region that included 28 consecutive A and T nucleotides and other A and T runs.

The average per-nucleotide error rate for two-pass sequencing was almost 0.6 percent, "with little variation as a function of read length," including only strands that were longer than 15 bases.

Over 98 percent of the more than 38,000 homopolymers of the BAC were covered with more than 10 reads and could thus be read, almost all of them correctly. Among homopolymers longer than 10 bases, fewer than half were covered with enough reads to call their length. "Increased sequencing depth, as is generated by the commercial instrument, would alleviate this problem," the authors wrote.

John Thompson, a Helicos researcher and senior author of the paper, told In Sequence via e-mail that the virtual terminators were included in Helicos' commercial sequencing kits from the beginning. "Helicos is constantly optimizing these and other single-molecule sequencing reagents for use in its commercial kits," he said.

Helicos said as early as 2007 that it had developed nucleotides that prevent the DNA polymerase from adding more than one base at a time, but provided scant details (see In Sequence 2/13/2007). According to Thompson, these previously described molecules were early versions of the current virtual terminator nucleotides.

Earlier this year, Helicos was issued a US patent, No. 7,476,734, which describes nucleotide analogs with a cleavable linker between the DNA base and the label.

Helicos is not the only company that has generated reversible terminator nucleotides with free 3' hydroxyl groups — LaserGen, a Houston-based start-up, has developed its own version (see In Sequence 3/3/2009).

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