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A hybrid approach for the automated finishing of bacterial genomes

Nature Biotechnology volume 30, pages 701–707 (2012)Cite this article

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Abstract

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.

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Figure 1: H1 assembly.
Figure 2: Resolution of rRNA genes.
Figure 3: CTX-TLC assembly and validation.
Figure 4: Superintegron assembly.

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NCBI Reference Sequence

Sequence Read Archive

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NCBI Reference Sequence

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Acknowledgements

This study was supported in part by the US National Institutes of Health National Institute of General Medical Sciences grant R01GM068851 (J.J.M. and W.P.R.), NIH R37 AI-42347 (B.M.D. and M.K.W.) and the Howard Hughes Medical Institute (B.M.D. and M.K.W.).

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Author notes

  1. Ali Bashir and Aaron A Klammer: These authors contributed equally to this work.

Authors and Affiliations

  1. Pacific Biosciences, Menlo Park, California, USA

    Ali Bashir, Aaron A Klammer, Chen-Shan Chin, Dale Webster, Ellen Paxinos, David Hsu, Meredith Ashby, Susana Wang, Paul Peluso, Robert Sebra, Jon Sorenson, James Bullard, Jackie Yen, Marie Valdovino, Emilia Mollova, Khai Luong, Steven Lin, Brianna LaMay, Amruta Joshi, Andrew Kasarskis & Eric E Schadt

  2. Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, USA

    Ali Bashir & Eric E Schadt

  3. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA

    William P Robins, John J Mekalanos & Matthew K Waldor

  4. National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

    Lori Rowe, Michael Frace, Cheryl L Tarr & Maryann Turnsek

  5. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Brigid M Davis & Matthew K Waldor

  6. Howard Hughes Medical Institute, Boston, Massachusetts, USA

    Brigid M Davis & Matthew K Waldor

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  1. Ali Bashir
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Contributions

A.B., A.A.K., W.P.R., C.S.C., E.P., M.F., C.L.T., M.T., B.M.D., A.K., J.J.M., M.K.W. and E.E.S. designed the experiments; A.B., A.A.K., C.S.C., D.W., J.S. and J.B. designed the methods; W.P.R., E.P., D.H., M.A., S.W., P.P., R.S., J.Y., M.V., E.M., K.L., S.L., B.L., A.J., L.R., M.F., C.L.T., M.T. and B.M.D. carried out all sample-preparation experiments, all sequencing runs and PCR-validation experiments; A.B., A.A.K., W.P.R., C.S.C., D.W., J.B., A.A.K., M.K.W. and E.E.S. jointly analyzed the data sets; and A.B., A.A.K., W.P.R., L.R., M.F., C.L.T., M.T., B.M.D., J.J.M., M.K.W. and E.E.S. wrote the manuscript.

Corresponding author

Correspondence to Eric E Schadt.

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Competing interests

Many of the authors are employees of and own stock in Pacific Biosciences.

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Supplementary Methods, Supplementary Results, Supplementary Tables 1-9 and Supplementary Figs. 1-13 (PDF 2063 kb)

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Bashir, A., Klammer, A., Robins, W. et al. A hybrid approach for the automated finishing of bacterial genomes. Nat Biotechnol 30, 701–707 (2012). https://doi.org/10.1038/nbt.2288

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