Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 M E T H O D O L O G Y A R T I C L E Open Access Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes Samuel O Oyola1*, Thomas D Otto1, Yong Gu1, Gareth Maslen1, Magnus Manske1, Susana Campino1, Daniel J Turner2, Bronwyn MacInnis1, Dominic P Kwiatkowski1, Harold P Swerdlow1 and Michael A Quail1 Abstract Background: Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. Results: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. Conclusion: We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material. Keywords: Next-Generation Sequencing, Illumina, Library, Plasmodium falciparum, AT-rich, Malaria, Clinical isolate, PCR, Tetramethyammonium chloride, PCR-free, Isothermal, Linear, Exponential Background Among the eukaryotic pathogens whose genomes have so far been sequenced, the malaria parasite, Plasmo- dium falciparum (P. falciparum), posed the most tech- nical challenges both in sequencing and assembly of the draft genome [1]. The biggest hurdle was the com- plexity of the genome, which is extremely base biased. * Correspondence: so1@sanger.ac.uk 1Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK Full list of author information is available at the end of the article The P. falciparum genome is very AT-rich; coding regions have a mean AT content of more than 75%, with up to 100% in intergenic and non-coding regions [2]. Although biased base composition has been a chal- lenge to the Sanger sequencing methods, it has contin- ued to be a major problem for the current high- throughput Next-Generation Sequencing (NGS) tech- nologies [3]. Much work has focused on solving ampli- fication and sequencing problems associated with high GC content but nothing has been done to improve on © 2012 Oyola et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.