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Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes - Nature Methods

  • ️Turner, Daniel J
  • ️Sun Mar 15 2009
  • Article
  • Published: 15 March 2009

Nature Methods volume 6, pages 291–295 (2009)Cite this article

Abstract

Amplification artifacts introduced during library preparation for the Illumina Genome Analyzer increase the likelihood that an appreciable proportion of these sequences will be duplicates and cause an uneven distribution of read coverage across the targeted sequencing regions. As a consequence, these unfavorable features result in difficulties in genome assembly and variation analysis from the short reads, particularly when the sequences are from genomes with base compositions at the extremes of high or low G+C content. Here we present an amplification-free method of library preparation, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly. We illustrate this by generating and analyzing DNA sequences from extremely (G+C)-poor (Plasmodium falciparum), (G+C)-neutral (Escherichia coli) and (G+C)-rich (Bordetella pertussis) genomes.

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Acknowledgements

This work was supported by the Wellcome Trust (grant WT079643). We thank C. Newbold and S. Kyes (University of Oxford) for providing DNA from P. falciparum 3D7.

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Author notes

  1. Iwanka Kozarewa and Zemin Ning: These authors contributed equally to this work.

Authors and Affiliations

  1. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    Iwanka Kozarewa, Zemin Ning, Michael A Quail, Mandy J Sanders, Matthew Berriman & Daniel J Turner

Authors

  1. Iwanka Kozarewa

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  2. Zemin Ning

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  3. Michael A Quail

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  4. Mandy J Sanders

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  5. Matthew Berriman

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  6. Daniel J Turner

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Contributions

I.K. planned and performed experiments; Z.N., M.J.S. and M.B. analyzed data; M.A.Q. prepared standard sequencing libraries; I.K. and D.J.T. devised the project; D.J.T., Z.N. and I.K. wrote the manuscript.

Corresponding author

Correspondence to Daniel J Turner.

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Kozarewa, I., Ning, Z., Quail, M. et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods 6, 291–295 (2009). https://doi.org/10.1038/nmeth.1311

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  • Received: 10 October 2008

  • Accepted: 17 February 2009

  • Published: 15 March 2009

  • Issue Date: April 2009

  • DOI: https://doi.org/10.1038/nmeth.1311