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Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

Nature Biotechnology volume 31, pages 11261132 (2013) | Download Citation

Abstract

Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing. MIDAS reduces amplification bias because polymerase cloning occurs in physically separated, nanoliter-scale reactors, facilitating the de novo assembly of near-complete microbial genomes from single Escherichia coli cells. In addition, MIDAS allowed us to detect single-copy number changes in primary human adult neurons at 1- to 2-Mb resolution. MIDAS can potentially further the characterization of genomic diversity in many heterogeneous cell populations.

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Acknowledgements

We thank C. Chen, H. Choi and the UCSD Nano3 facility for initial help with microwell fabrication, F. Liang for initial technical assistance and P. Pevzner for advice on de novo genome assembly. This project was funded by US National Institutes of Health grants R01HG004876, R01GM097253, U01MH098977 and P50HG005550, and National Science Foundation grant OCE-1046368.

Author information

Author notes

    • Hsin-I Chiang

    Present address: Department of Animal Science, National Chung Hsing University, Taichung, Taiwan.

Affiliations

  1. Department of Bioengineering, Institute for Genomic Medicine and Institute of Engineering in Medicine, University of California at San Diego, La Jolla, California, USA.

    • Jeff Gole
    • , Athurva Gore
    • , Andrew Richards
    • , Ho-Lim Fung
    • , Hsin-I Chiang
    •  & Kun Zhang
  2. Materials Science and Engineering Program, University of California at San Diego, La Jolla, California, USA.

    • Yu-Jui Chiu
  3. Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California, USA.

    • Diane Bushman
    •  & Jerold Chun
  4. Department of Electrical and Computer Engineering, University of California at San Diego, La Jolla, California, USA.

    • Yu-Hwa Lo

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Contributions

J.G. and K.Z. conceived and designed the experiments. J.G., A.R. and H.-I.C. performed the experiments. J.G. and Y.-J.C. fabricated the microwell arrays. H.-L.F. performed sequencing. D.B. provided neuronal nuclei. J.G., A.G. and K.Z. analyzed data and wrote the manuscript with input from Y.-H.L. and J.C.

Competing interests

J.G. and K.Z. are listed as co-inventors in a patent application related to this work.

Corresponding author

Correspondence to Kun Zhang.

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DOI

https://doi.org/10.1038/nbt.2720