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Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data

Nature Methods volume 10, pages 563–569 (2013)Cite this article

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Abstract

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph–based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

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Figure 1: Principle of the hierarchical genome-assembly process using long-insert-size DNA shotgun template libraries with SMRT sequencing.
Figure 2: Workflow for the de novo HGAP assembly of E. coli MG1655.

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Sequence Read Archive

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NCBI Reference Sequence

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Acknowledgements

We thank S. Clingenpeel (Joint Genome Institute) for growing cultures and performing DNA extraction for M. ruber and P. heparinus; B. Munson and F. Antonacci for assistance with the BAC library construction; and K. Travers, S. McCalmon, M. Wang, U. Nguyen, S. Ranade, M. Ashby, L. Hon and L. Hickey (Pacific Biosciences) for assistance in sample preparation, sequencing and data analysis. The authors acknowledge the ATCC for providing the E. coli K-12 MG1655 strain. We thank S. Koren and A. Phillippy for pointing out to us the SMRT sequencing–based gap-filling functionality development in the Celera Assembler. The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231.

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Authors and Affiliations

  1. Pacific Biosciences, Menlo Park, California, USA

    Chen-Shan Chin, David H Alexander, Patrick Marks, Aaron A Klammer, James Drake, Cheryl Heiner, Stephen W Turner & Jonas Korlach

  2. Joint Genome Institute, Walnut Creek, California, USA

    Alicia Clum & Alex Copeland

  3. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    John Huddleston & Evan E Eichler

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  1. Chen-Shan Chin
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Contributions

C.-S.C., A. Copeland., E.E.E., S.W.T. & J.K. designed the experiments; C.-S.C., D.H.A., P.M., A.A.K., J.D., A. Clum and J.H. analyzed data; C.H. performed the validation sequencing; and C.-S.C., D.H.A., P.M., A.A.K., A. Copeland., E.E.E. and J.K. wrote the manuscript.

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Correspondence to Jonas Korlach.

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Competing interests

C.-S.C., D.H.A., P.M., A.A.K., J.D., C.H., S.W.T. and J.K. are employees of Pacific Biosciences, a company commercializing DNA sequencing technologies.

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Supplementary Figures 1–15, Supplementary Tables 1–5 and Supplementary Notes 1 and 2 (PDF 3942 kb)

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Chin, CS., Alexander, D., Marks, P. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10, 563–569 (2013). https://doi.org/10.1038/nmeth.2474

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