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Long-range polony haplotyping of individual human chromosome molecules

Nature Genetics volume 38, pages 382–387 (2006)Cite this article

Abstract

We report a method for multilocus long-range haplotyping on human chromosome molecules in vitro based on the DNA polymerase colony (polony) technology. By immobilizing thousands of intact chromosome molecules within a polyacrylamide gel on a microscope slide and performing multiple amplifications from single molecules, we determined long-range haplotypes spanning a 153-Mb region of human chromosome 7 and found evidence of rare mitotic recombination events in human lymphocytes. Furthermore, the parallel nature of DNA polony technology allows efficient haplotyping on pooled DNAs from a population on one slide, with a throughput three orders of magnitudes higher than current molecular haplotyping methods. Linkage disequilibrium statistics established by our pooled DNA haplotyping method are more accurate than statistically inferred haplotypes. This haplotyping method is well suited for candidate gene–based association studies as well as for investigating the pattern of recombination in mammalian cells.

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Figure 1: Multi-locus long-range haplotyping.
Figure 2: Haplotyping on intact chromosomes.

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References

  1. Michalatos-Beloin, S., Tishkoff, S.A., Bentley, K.L., Kidd, K.K. & Ruano, G. Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR. Nucleic Acids Res. 24, 4841–4843 (1996).

    Article  CAS  Google Scholar 

  2. Ding, C. & Cantor, C.R. Direct molecular haplotyping of long-range genomic DNA with M1-PCR. Proc. Natl. Acad. Sci. USA 100, 7449–7453 (2003).

    Article  CAS  Google Scholar 

  3. Mitra, R.D. et al. Digital genotyping and haplotyping with polymerase colonies. Proc. Natl. Acad. Sci. USA 100, 5926–5931 (2003).

    Article  CAS  Google Scholar 

  4. Woolley, A.T., Guillemette, C., Li Cheung, C., Housman, D.E. & Lieber, C.M. Direct haplotyping of kilobase-size DNA using carbon nanotube probes. Nat. Biotechnol. 18, 760–763 (2000).

    Article  CAS  Google Scholar 

  5. Douglas, J.A., Boehnke, M., Gillanders, E., Trent, J.M. & Gruber, S.B. Experimentally-derived haplotypes substantially increase the efficiency of linkage disequilibrium studies. Nat. Genet. 28, 361–364 (2001).

    Article  CAS  Google Scholar 

  6. Patil, N. et al. Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21. Science 294, 1719–1723 (2001).

    Article  CAS  Google Scholar 

  7. Mitra, R.D. & Church, G.M. In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. 27, e34 (1999).

    Article  CAS  Google Scholar 

  8. Zhu, J., Shendure, J., Mitra, R.D. & Church, G.M. Single molecule profiling of alternative pre-mRNA splicing. Science 301, 836–838 (2003).

    Article  CAS  Google Scholar 

  9. Mitra, R.D., Shendure, J., Olejnik, J., Edyta Krzymanska, O. & Church, G.M. Fluorescent in situ sequencing on polymerase colonies. Anal. Biochem. 320, 55–65 (2003).

    Article  CAS  Google Scholar 

  10. SantaLucia, J., Jr. & Hicks, D. The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct. 33, 415–440 (2004).

    Article  CAS  Google Scholar 

  11. Stephens, M., Smith, N.J. & Donnelly, P. A new statistical method for haplotype reconstruction from population data. Am. J. Hum. Genet. 68, 978–989 (2001).

    Article  CAS  Google Scholar 

  12. Tishkoff, S.A., Pakstis, A.J., Ruano, G. & Kidd, K.K. The accuracy of statistical methods for estimation of haplotype frequencies: an example from the CD4 locus. Am. J. Hum. Genet. 67, 518–522 (2000).

    Article  CAS  Google Scholar 

  13. Cohen, J.C. et al. Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Science 305, 869–872 (2004).

    Article  CAS  Google Scholar 

  14. Wang, N., Akey, J.M., Zhang, K., Chakraborty, R. & Jin, L. Distribution of recombination crossovers and the origin of haplotype blocks: the interplay of population history, recombination, and mutation. Am. J. Hum. Genet. 71, 1227–1234 (2002).

    Article  CAS  Google Scholar 

  15. Forton, J. et al. Accuracy of haplotype reconstruction from haplotype-tagging single-nucleotide polymorphisms. Am. J. Hum. Genet. 76, 438–448 (2005).

    Article  CAS  Google Scholar 

  16. Patterson, N. et al. Methods for high-density admixture mapping of disease genes. Am. J. Hum. Genet. 74, 979–1000 (2004).

    Article  CAS  Google Scholar 

  17. Smith, M.W. et al. A high-density admixture map for disease gene discovery in african americans. Am. J. Hum. Genet. 74, 1001–1013 (2004).

    Article  CAS  Google Scholar 

  18. Bersaglieri, T. et al. Genetic signatures of strong recent positive selection at the lactase gene. Am. J. Hum. Genet. 74, 1111–1120 (2004).

    Article  CAS  Google Scholar 

  19. Prado, F., Cortes-Ledesma, F., Huertas, P. & Aguilera, A. Mitotic recombination in Saccharomyces cerevisiae. Curr. Genet. 42, 185–198 (2003).

    CAS  PubMed  Google Scholar 

  20. Hendricks, C.A. et al. Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice. Proc. Natl. Acad. Sci. USA 100, 6325–6330 (2003).

    Article  CAS  Google Scholar 

  21. Meyn, M.S. High spontaneous intrachromosomal recombination rates in ataxia-telangiectasia. Science 260, 1327–1330 (1993).

    Article  CAS  Google Scholar 

  22. Kehrer-Sawatzki, H. et al. High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene. Am. J. Hum. Genet. 75, 410–423 (2004).

    Article  CAS  Google Scholar 

  23. Winckler, W. et al. Comparison of fine-scale recombination rates in humans and chimpanzees. Science 308, 107–111 (2005).

    Article  CAS  Google Scholar 

  24. Jeffreys, A.J., Neumann, R., Panayi, M., Myers, S. & Donnelly, P. Human recombination hot spots hidden in regions of strong marker association. Nat. Genet. 37, 601–606 (2005).

    Article  CAS  Google Scholar 

  25. Kauppi, L., Jeffreys, A.J. & Keeney, S. Where the crossovers are: recombination distributions in mammals. Nat. Rev. Genet. 5, 413–424 (2004).

    Article  CAS  Google Scholar 

  26. von Ahsen, N., Wittwer, C.T. & Schutz, E. Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin. Chem. 47, 1956–1961 (2001).

    CAS  PubMed  Google Scholar 

  27. Cram, L.S., Bell, C.S. & Fawcett, J.J. Chromosome sorting and genomics. Methods Cell Sci. 24, 27–35 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank N. Reppas, F. Isaacs, J. Akey and L. Jin for critical review of the manuscript, C. Varma for assistance in polony image analyses. This work was supported by the US National Human Genome Research Institute-Center of Excellence in Genome Science grants.

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Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Kun Zhang, Jay Shendure, Gregory J Porreca, John D Aach & George M Church

  2. Institute of Genome Science and Policy, Duke University, Durham, 27708, North Carolina, USA

    Jun Zhu

  3. Department of Genetics, Washington University, St. Louis, 63108, Missouri, USA

    Robi D Mitra

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  1. Kun Zhang
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  2. Jun Zhu
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  7. George M Church
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Correspondence to Kun Zhang.

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Supplementary information

Supplementary Fig. 1

Multi-locus polony haplotyping on CD36 and NOS3. (PDF 134 kb)

Supplementary Fig. 2

Effects of sample size on LD patterns revealed by pooled polony haplotyping. (PDF 164 kb)

Supplementary Table 1

Summary statistics of polony haplotyping on pooled DNA of 24 African Americans. (PDF 50 kb)

Supplementary Table 2

CD36 haplotypes determined by polony haplotyping. (PDF 31 kb)

Supplementary Table 3

Comparison of LD statistics in NOS3 determined by four methods. (PDF 54 kb)

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Zhang, K., Zhu, J., Shendure, J. et al. Long-range polony haplotyping of individual human chromosome molecules. Nat Genet 38, 382–387 (2006). https://doi.org/10.1038/ng1741

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