Parallel, tag-directed assembly of locally derived short sequence reads

Abstract

We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.

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Figure 1: Schematic of subassembly process.
Figure 2: Evaluation of subassembly performance.

References

  1. 1

    Shendure, J. & Ji, H. Nat. Biotechnol. 26, 1135–1145 (2008).

    CAS  Article  Google Scholar 

  2. 2

    Hillier, L.W. et al. Nat. Methods 5, 183–188 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Nat. Methods 5, 621–628 (2008).

    CAS  Article  Google Scholar 

  4. 4

    Hamady, M. & Knight, R. Genome Res. 19, 1141–1152 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Simpson, J.T. et al. Genome Res. 19, 1117–1123 (2009).

    CAS  Article  Google Scholar 

  6. 6

    Weinstein, J.A., Jiang, N., White, R.A. 3rd, Fisher, D.S. & Quake, S.R. Science 324, 807–810 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Bentley, G. et al. Tissue Antigens 74, 393–403 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Margulies, M. et al. Nature 437, 376–380 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Kalyuzhnaya, M.G. et al. Nat. Biotechnol. 26, 1029–1034 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Ewing, B. & Green, P. Genome Res. 8, 186–194 (1998).

    CAS  Article  Google Scholar 

  11. 11

    Stover, C.K. et al. Nature 406, 959–964 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Altschul, S.F. et al. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

  13. 13

    Myers, E.W. et al. Science 287, 2196–2204 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Reinhardt, J.A. et al. Genome Res. 19, 294–305 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Zerbino, D.R. & Birney, E. Genome Res. 18, 821–829 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Brady, A. & Salzberg, S.L. Nat. Methods 6, 673–676 (2009).

    CAS  Article  Google Scholar 

  17. 17

    Delcher, A.L., Harmon, D., Kasif, S., White, O. & Salzberg, S.L. Nucleic Acids Res. 27, 4636–4641 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

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Acknowledgements

We thank L. Chistoserdova and M.G. Kalyuzhnaya (University of Washington) for the gift of the methylamine-enriched metagenomic DNA sample, C. Manoil (University of Washington) for the gift of P. aeruginosa strain PAO1 genomic DNA and P. Green for helpful discussions. J.B.H. is supported by US National Institutes of Health grant T32GM007266 and an Achievement Rewards for College Scientists fellowship.

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Contributions

E.H.T. and J.S. conceived the initial approach. All authors contributed to subsequent experimental design. J.B.H. and E.H.T. developed library construction methods. C.L. performed Illumina sequencing. R.P.P. developed the subassembly computational pipeline and iterative scaffolding algorithm. J.B.H., R.P.P. and J.S. analyzed data. All authors contributed to writing of the manuscript. J.S. supervised all aspects of the study.

Corresponding authors

Correspondence to Joseph B Hiatt or Jay Shendure.

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Competing interests

J.S., J.B.H., R.P.P. and E.H.T. are authors of a patent application for the method described in this paper (US Provisional Application number 61/096,720).

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Supplementary Figures 1–6, Supplementary Tables 1–4, Supplementary Notes 1–4 and Supplementary Protocols 1–3 (PDF 837 kb)

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Hiatt, J., Patwardhan, R., Turner, E. et al. Parallel, tag-directed assembly of locally derived short sequence reads. Nat Methods 7, 119–122 (2010). https://doi.org/10.1038/nmeth.1416

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