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.
Next-generation sequencing Amplification-free library preparation Malaria (A + T)-rich genome
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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. Nature456, 53 – 59.PubMedCrossRefGoogle Scholar
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. BioTechniques25, 684– 691.PubMedGoogle Scholar
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 Testing2, 351–355.PubMedCrossRefGoogle Scholar
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. Nature419, 498– 511.PubMedCrossRefGoogle Scholar
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 Methods6, 291– 295.PubMedCrossRefGoogle Scholar