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. 2009 Aug;6(8):593-5.
doi: 10.1038/nmeth.1354. Epub 2009 Jul 20.

Virtual terminator nucleotides for next-generation DNA sequencing

Jayson Bowers  1 , Judith Mitchell, Eric Beer, Philip R Buzby, Marie Causey, J William Efcavitch, Mirna Jarosz, Edyta Krzymanska-Olejnik, Li Kung, Doron Lipson, Geoffrey M Lowman, Subramanian Marappan, Peter McInerney, Adam Platt, Atanu Roy, Suhaib M Siddiqi, Kathleen Steinmann, John F Thompson
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Virtual terminator nucleotides for next-generation DNA sequencing

Jayson Bowers (V体育ios版) et al. Nat Methods. 2009 Aug.

VSports - Abstract

We synthesized reversible terminators with tethered inhibitors for next-generation sequencing VSports手机版. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them 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. .

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Figures

Figure 1
Figure 1. Virtual Terminator nucleotide base-by-base incorporation in a G5 homopolymer
The substrate used for testing homopolymer sequencing is shown along with successive cycles of addition of compound 22 in a solution phase reaction. Removal of the inhibitor-dye was accomplished by cleavage of the disulfide using TCEP, a reducing agent, followed by treatment with iodoacetamide to cap the free thiol. After each cycle, an aliquot of the reaction is run on an ABI3730 sequencing machine to achieve single base resolution of DNA. Length markers are shown in orange and the DNA being synthesized is shown in blue.
Figure 2
Figure 2. Sequence coverage of BAC
Depth of coverage for the BAC is shown across its length. When all reads (non-unique) were mapped to the sequence (red), repeat regions received higher coverage due to multiply aligning reads. When only uniquely aligning sequences were included (blue), the repeat regions were under-represented. When a fractional correction was made to multiply aligning sequences (black), even coverage was obtained across the length of the BAC. When only unique alignments were used, the longest uncovered stretch of DNA was 279 bp, corresponding to a repeat region. If all alignments were used, the longest uncovered region was a highly AT rich 103 bp segment that included 28 consecutive AT nucleotides and many other shorter AT runs.
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