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Helicos Scientists, Collaborators Sequence Viral Genome with Single-Molecule Sequencing Method

NEW YORK (GenomeWeb News) – Scientists from Helicos BioSciences, Ohio University, and Stanford University today published a paper describing the first single-molecule sequencing of a whole genome.
The researchers used a single-molecule sequencing, sequencing-by-synthesis method, developed by Helicos, to sequence the roughly 7,000-nucleotide genome of the M13 virus. The work appears online today in the journal Science.
In the company's version of single-molecule sequencing, an approach first proposed in the late 1980s, nucleic acid templates that are created by digesting genomic DNA are hybridized to primers that are covalently anchored in random positions on a proprietary glass cover slip in a flow cell.
Then, a polymerase and labeled DNA bases are added, one nucleotide at a time. After they are incorporated into a complementary strand, these labeled bases are detected by fluorescence imaging.
Because a signal is detected from a single DNA molecule, the method obviates the need for amplification by cloning or PCR. And, the researchers say, the availability of a consensus human genome sequence is changing the sequencing landscape.
“[O]ne can use low-cost techniques with much shorter read lengths and higher parallelism than found with the Sanger capillary electrophoresis methods used to generate the reference genome,” lead author Timothy Harris, senior director of research at Helicos BioSciences, and his colleagues wrote.
For this study, the team used single-molecule sequencing on more than 280,000 primer-template duplexes at once, sequencing the genome of M13, a virus that normally infects Enterobacteria. In total, the M13 genomic DNA went through 224 sequencing cycles.
The approach reportedly enabled 100 percent coverage of the double-stranded DNA form of the phage’s 6,407 nucleotide genome by sequencing to a depth of more than 150-fold. Read lengths averaged about 23 bases, though the authors noted that they could increase their read lengths by using more cycles.
Harris and his team re-sequenced the genome using high-fidelity polymerase. They sequenced each individual strand twice and calculated a “two-pass error rate.” About 80 percent of the bases were in agreement from both passes. When errors did occur, most of them were deletions.
The authors noted that there were still some limitations to the single-molecule technology that they used in the study, which was based on the "first generation" of the company's single molecule chemistry. For instance, they found that in a run of bases of the same kind, multiple fluorophore incorporations could decrease emissions, sometimes below the level of detection. Still, they said, the method could bring researchers closer to affordable, high-throughput, comparative sequencing of thousands of human genomes.
Bill Efcavitch, senior vice president for product R&D at Helicos, said in a statement that the company has "since developed new generations of 'one-base-at-a-time' nucleotides which allow more accurate homopolymer sequencing, and lower overall error rates."
The authors of the study conclude that “the simplicity of the methods described here, the freedom from cloning or amplification, and the low reagent volumes used to produce sequence from over 280,000 strands simultaneously opens a path to very high throughput sequencing.”
The company declined to comment further on the study.

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