Improvement of PCR-free NGS Library Preparation to Obtain Uniform Read Coverage of Genome with Extremely High AT Content

摘要

PCR amplification is commonly used in generating libraries for Next-Generation Sequencing (NGS) to efficiently enrich and amplify sequenceable DNA fragments. However, it introduces bias in the representation of the original complex template DNA. Such artifact has devastating effects in sequencing genomes with highly unbalanced base composition: regions of extremely high or low GC content, which are a substantial fraction of such genomes, are often covered with zero or near-zero read depth. PCR-free library preparation method has been published, which utilizes quantitative PCR and relies on previously sequenced similar libraries as standards to measure the amount of properly ligated fragments to obtain the library molecular concentration required by the sequencer. Here we present improvements of the PCR-free library preparation method to assess the efficiency of generating sequenceable library fragments and omit the quantitative PCR step. This is achieved by quantifying the ratio between properly ligated library fragments and improperly ligated fragments based on their fragment size difference and making use of the recently released Illumina TruSeq DNA Sample Prep kit that accommodates PCR-free library generation. Our improvement was applied to the highly AT-rich malaria parasite, Plasmodium falciparum, and the sequencing yield from Illumina HiSeq 2000 was optimal. Compared to the library generated with PCR amplification, our improvement increased the coverage uniformity across the whole P. falciparum genome of over 80% AT in a similar way as the published PCR-free method. Further enhancement is under our way to lower the input DNA amount to make the PCR-free library preparation method more widely applicable in genomic research. Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities.