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Decoding long nanopore sequencing reads of natural DNA

Nature Biotechnology volume 32, pages 829–833 (2014)Cite this article

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Abstract

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

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Figure 1: Experimental schematic and raw data.
Figure 2: A quadromer map predicts current levels for previously unmeasured DNA.
Figure 3: Raw data to alignment.
Figure 4: Alignments to reference sequence and hybrid reconstruction.

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Acknowledgements

Thanks to J.J. Bartlett and B. Tickman for their help in data acquisition. This work was supported by the National Institutes of Health, National Human Genome Research Institutes (NHGRI) $1,000 Genome Program Grants R01HG005115, R01HG006321 and R01HG006283 and graduate research fellowship DGE-0718124 from the National Science Foundation (to A.A.).

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Authors and Affiliations

  1. Department of Physics, University of Washington, Seattle, Washington, USA

    Andrew H Laszlo, Ian M Derrington, Brian C Ross, Henry Brinkerhoff, Ian C Nova, Jonathan M Craig, Kyle W Langford, Jenny Mae Samson, Kenji Doering & Jens H Gundlach

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

    Andrew Adey, Riza Daza & Jay Shendure

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  1. Andrew H Laszlo
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Contributions

A.H.L., I.M.D., A.A., J.S. and J.H.G. designed the research. H.B., A.A., I.C.N., J.M.C., J.M.S., R.D. and K.D. performed the research. A.H.L., I.M.D., B.C.R., H.B., I.C.N., J.M.C. and K.W.L. analyzed the data. A.H.L., J.H.G. and J.S. wrote the paper.

Corresponding author

Correspondence to Jens H Gundlach.

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Competing interests

I.M.D. has a commercial interest with Illumina Inc. The University of Washington has filed a provisional patent on technologies described herein.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Notes 1–5 and Supplementary Tables 1–6 (PDF 4418 kb)

Supplementary Software

Software code. (ZIP 22513 kb)

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Laszlo, A., Derrington, I., Ross, B. et al. Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol 32, 829–833 (2014). https://doi.org/10.1038/nbt.2950

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