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Single-molecule sequencing of an individual human genome

Abstract

Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28× average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.

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Figure 1: P0 genome sequencing metrics.
Figure 2: SNP discovery in P0.
Figure 3: Copy number variation in the P0 human genome.

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References

  1. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    CAS  Article  Google Scholar 

  2. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    CAS  Article  Google Scholar 

  3. Levy, S. et al. The diploid genome sequence of an individual human. PLoS Biol. 5, e254 (2007).

    Article  Google Scholar 

  4. Wheeler, D.A. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876 (2008).

    CAS  Article  Google Scholar 

  5. Ley, T.J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008).

    CAS  Article  Google Scholar 

  6. Wang, J. et al. The diploid genome sequence of an Asian individual. Nature 456, 60–65 (2008).

    CAS  Article  Google Scholar 

  7. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    CAS  Article  Google Scholar 

  8. Kim, J.I. et al. A highly annotated whole-genome sequence of a Korean individual. Nature advance online publication doi:10.1038/nature08211 (8 July 2009).

  9. Ahn, S.-M. et al. The first Korean genome sequence and analysis: full genome sequencing for a socio-ethnic group. Genome Res. published online, doi:10.1101/gr.092197.109 (26 May 2009).

  10. Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S.R. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA 100, 3960–3964 (2003).

    CAS  Article  Google Scholar 

  11. Greenleaf, W.J. & Block, S.M. Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 313, 801 (2006).

    CAS  Article  Google Scholar 

  12. Harris, T.D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).

    CAS  Article  Google Scholar 

  13. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    CAS  Article  Google Scholar 

  14. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    CAS  Article  Google Scholar 

  15. Rumble, S.M. et al. SHRiMP: Accurate Mapping of Short Color-space Reads. PLOS Comput. Biol. 5, 1000386 (2009).

    Article  Google Scholar 

  16. Daines, B. et al. High-throughput multiplex sequencing to discover copy number variants in Drosophila. Genetics published online, doi:10.1534/genetics.109.103218 (15 June 2009).

  17. Herman, D.S. et al. Filter-based hybridization capture of subgenomes enables resequencing and copy-number detection. Nat. Methods 6, 507–510 (2009).

    CAS  Article  Google Scholar 

  18. Xie, C. & Tammi, M.T. CNV-seq, a new method to detect copy number variation using high-throughput sequencing. BMC Bioinformatics 10, 80 (2009).

    Article  Google Scholar 

  19. Iafrate, A.J. et al. Detection of large-scale variation in the human genome. Nat. Genet. 36, 949–951 (2004).

    CAS  Article  Google Scholar 

  20. Qin, J., Jones, R.C. & Ramakrishnan, R. Studying copy number variations using a nanofluidic platform. Nucleic Acids Res. 36, e116 (2008).

    Article  Google Scholar 

  21. Vijg, J., Busuttil, R.A., Bahar, R. & Dollé, M.E. Aging and genome maintenance. Ann. NY Acad. Sci. 1055, 35–47 (2005).

    CAS  Article  Google Scholar 

  22. Janeway, C. Immunobiology (Garland Science, New York, 2004).

    Google Scholar 

Download references

Acknowledgements

We are grateful to V. Natu and J. Coller of the Stanford Functional Genomics Facility for performing the Illumina SNP analysis, R.A. White for assistance with dPCR assays, and A. Sidow for the use of the Covaris sonicator. We acknowledge National Science Foundation award CNS-0619926 for computer resources funding the Bio-X2 cluster, and the National Institutes of Health Pioneer Award (to S.R.Q.).

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Authors

Contributions

N.F.N. prepared the libraries, performed the sequencing and wrote the manuscripts. D.P. developed the data analysis algorithms, performed the computations and wrote the manuscript. S.R.Q. designed the research and wrote the manuscript.

Corresponding author

Correspondence to Stephen R Quake.

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Competing interests

D.P. owns shares of Helicos. S.R.Q. is a founder, shareholder and consultant for Helicos and Fluidigm.

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Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 126 kb)

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Pushkarev, D., Neff, N. & Quake, S. Single-molecule sequencing of an individual human genome. Nat Biotechnol 27, 847–850 (2009). https://doi.org/10.1038/nbt.1561

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