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Rapid genome sequencing with short universal tiling probes


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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Figure 1: A massively parallel DNA display platform based on in situ RCA.
Figure 2: Probe design and characterization.
Figure 3: Fragments aligned to the reference genome in the Bacteriophage λ assembly.
Figure 4: Probabilistic basecalling algorithm.
Figure 5: The depth of coverage along the E. coli chromosome was strongly skewed toward the origin of replication.
Figure 6: Assembly statistics for the E. coli genome.

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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.

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Authors and Affiliations



A.P. developed the short LNA probes, participated in the development of sample preparation, the rolling-circle arrays and the hybridization cycle. G.B. participated in the development of sample preparation and the hybridization cycle. A.M. participated in the development of sample preparation and the rolling-circle arrays. P.L. participated in the development of image analysis and basecalling software and algorithms. E.H. developed the custom Peltier assembly, built the instrument and participated in the development of instrument control and image analysis software. S.L. conceived of the concept of shotgun SBH, participated in probe design, the development of sample preparation, rolling-circle arrays, hybridization cycle, and of the instrument control, image analysis and basecalling software; analyzed the experiments, directed the research and drafted the manuscript.

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Correspondence to Sten Linnarsson.

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Competing interests

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).

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