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Single-molecule sequencing of an individual human genome

Nature Biotechnology volume 27, pages 847–850 (2009)Cite this article

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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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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Author notes

  1. Dmitry Pushkarev and Norma F Neff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA

    Dmitry Pushkarev, Norma F Neff & Stephen R Quake

Authors
  1. Dmitry Pushkarev
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  2. Norma F Neff
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  3. Stephen R Quake
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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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