JF: Virtual terminator nucleotides for next-generation DNA sequencing. Nat Methods 2009

We synthesized reversible terminators with tethered inhibitors for next generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides and were used to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99 % of the region sequenced. This single-molecule approach generated high quality sequence data without the need for target amplification and thus avoided concomitant biases. Highly parallel sequencing technologies have revolutionized biology by providing orders of magnitude more DNA sequence data than previously possible1. Most of these technologies require synthesizing DNA from an existing template using either a polymerase or ligase2. The first commercialized technologies required amplification of the template DNA prior to sequencing, but this can introduce a host of biases caused by differential behavior from many factors3, making even representation or accurate quantitation of samples difficult. Single-molecule sequencing4,5 can eliminate biases introduced by amplification. Any method employing polymerase-based sequencing-by-synthesis encounters the proble