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Amplification-Free Library Preparation for Paired-End Illumina Sequencing

  • Iwanka Kozarewa
  • Daniel J. TurnerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 733)

Abstract

The library preparation step is of critical importance for the quality of next-generation sequencing data. The use of the polymerase chain reaction (PCR) as a part of the standard Illumina library preparation protocol causes an appreciable proportion of the obtained sequences to be duplicates, making the sequencing run less efficient. Also, amplification introduces biases, particularly for genomes with high or low GC content, which reduces the complexity of the resulting library. To overcome these difficulties, we developed an amplification-free library preparation. By the use of custom adapters, unamplified, ligated samples can hybridize directly to the oligonucleotides on the flowcell surface.

Key words

Next-generation sequencing Amplification-free library preparation Malaria (A  +  T)-rich genome 

References

  1. 1.
    Bentley D. R., Balasubramanian S., Swerdlow H. P., Smith G. P., Milton J., Brown C. G. et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53  –  59.PubMedCrossRefGoogle Scholar
  2. 2.
    Barnard R., Futo V., Pecheniuk N., Slattery M. and Walsh T. (1998) PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis. BioTechniques 25, 684–  691.PubMedGoogle Scholar
  3. 3.
    Hahn S., Garvin A. M., Di Naro E. and Holzgreve W. (1998) Allele drop-out can occur in alleles differing by a single nucleotide and is not alleviated by preamplification or minor template increments. Genetic Testing 2, 351–355.PubMedCrossRefGoogle Scholar
  4. 4.
    Gardner M. J., Hall N., Fung E., White O., Berriman M., Hyman R. W. et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–  511.PubMedCrossRefGoogle Scholar
  5. 5.
    Kozarewa I., Ning Z., Quail M. A., Sanders M. J., Berriman M. and Turner D. J. (2009) Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nature Methods 6, 291–  295.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.The Wellcome Trust Sanger InstituteCambridgeUK

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