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

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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Figure 1: No-PCR library preparation.
Figure 2: Distribution of genome sequence coverage across the unmasked genomes for various datasets with or without the PCR step.
Figure 3: Distribution of sequenced reads for different values of G+C content.
Figure 4: Frequencies of duplicate sequences.

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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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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.

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Correspondence to Daniel J Turner.

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Supplementary Figures 1–2, Supplementary Table 1, Supplementary Methods (PDF 1188 kb)

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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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