The increasing availability of high-quality reference genomic sequences has created a demand for ways to survey the sequence differences present in individual genomes. Here we describe a DNA sequencing method based on hybridization of a universal panel of tiling probes. Millions of shotgun fragments are amplified in situ and subjected to sequential hybridization with short fluorescent probes. Long fragments of 200 bp facilitate unique placement even in large genomes. The sequencing chemistry is simple, enzyme-free and consumes only dilute solutions of the probes, resulting in reduced sequencing cost and substantially increased speed. A prototype instrument based on commonly available equipment was used to resequence the Bacteriophage λ and Escherichia coli genomes to better than 99.93% accuracy with a raw throughput of 320 Mbp/day, albeit with a significant number of small gaps attributed to losses in sample preparation.
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We thank M. Nilsson and U. Landegren for early advice on RCA; J. Sagemark for initial bioinformatics analysis of the feasibility of the concept; P. Bérubé for the E. coli DNA preparation; M. Belouchi, P. van Eerdewegh, J. Hooper, B. Houle, R. Paulussen for helpful discussions; and P. Ernfors for advice and discussions. This work was supported by Swedish Research Council grant 522-2006-6511.
The authors are former employees of Genizon Svenska AB, an affiliate of Genizon Biosciences Inc. (Montreal), which has also funded the research. The authors may under certain circumstances stand to receive royalty payments or similar benefits related to the technology presented in the paper.
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Pihlak, A., Baurén, G., Hersoug, E. et al. Rapid genome sequencing with short universal tiling probes. Nat Biotechnol 26, 676–684 (2008). https://doi.org/10.1038/nbt1405
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