Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 show a significant reduction in base-composition bias, this strategy requires large quantities of starting material. Therefore, PCR-free library preparation is unsuitable where amounts of DNA starting material are limited, as is the case with most clinical isolates. We demonstrate that problems associated with PCR can be ameliorated through optimization, thereby allow- ing amplification to be used in generating sequencing libraries even from extremely AT-biased genomes. Through careful selection of polymerases and the use of a PCR-additive that normalizes melting temperatures of the different library fragments, we have optimized Illu- mina library amplification. These alternative PCR condi- tions generate library fragments with increased coverage of extreme AT-rich regions. Standard Illumina amplification procedures that use Phusion polymerase generate libraries with biased base- composition and are depleted of AT-rich loci. The sequence data generated under these standard conditions are of reduced complexity and maybe unsuitable for rese- quencing to identify relatively rare genetic variants of important biological function. We set out to solve this problem by focusing on the library amplification step, which is the primary source of biases in almost all NGS platforms. We started by screening commercially avail- able polymerases for tolerance to an extremely AT-rich template. This initial screening procedure identified a preferred polymerase and amplification conditions that showed improved tolerance to AT-rich templates. We demonstrated that most commercial polymerases are inhibited by TMAC. However, this chemical can have a tremendous positive effect in amplifying AT-rich tem- plates for the TMAC-tolerant enzymes. With TMAC additive we have developed Illumina library-amplification protocol suitable for extreme AT-rich genomic DNA. Quality analyses of sequence data from both P. falci- parum 3D7 and a clinical isolate generated by our opti- mized conditions showed less bias towards base-balanced regions and produced an average GC content that matches that of the unamplified genome. To date, no single polymerase or amplification method has been shown to efficiently amplify both extremes of base composition with equal and optimum efficiency. The two isothermal amplification methods (RPA and T7) tested here showed some level of improved coverage rela- tive to standard amplifications, but they were both asso- ciated with high levels of chimeric and duplicate reads. Under the optimized conditions we show that the Kapa series of polymerases in the presence of TMAC produced the best coverage that was relatively consistent across regions of extreme base composition. Worthy of note is the low template-dependent fidelity score for Kapa HiFi (Table 2), where we detected a number of false-positive insertion calls mainly in regions of TATA repeats. Other Page 8 of 12 enzymes had zero or extremely low coverage in these regions, limiting our ability to perform accurate com- parative analysis on enzyme’s fidelity at very low com- plexity regions. Conclusion Despite the bias problem associated with it, PCR still remains an essential step in many NGS library prepara- tion procedures, especially when the starting DNA mate- rial is limited. We describe robust alternative PCR conditions optimized for extremely AT-biased genomes. We show that the optimized conditions significantly reduce bias and also retain the complexity of either extremes of base composition. This observation was con- sistent in both the lab-cultured strain (3D7) and the clini- cal isolate. The optimized procedures described here will greatly benefit the malaria genomic research and control initiatives that have faced serious challenges with the parasite’s extreme AT-rich genome. Methods Genomic DNA P. falciparum 3D7 genomic DNA was a gift from Prof Chris Newbold (University of Oxford). Other P. falci- parum genomic DNA (PK0076) was a clinical isolate containing ~53% human DNA contamination. The clini- cal isolate was obtained from the Malaria Genetics Group’s Sequencing Sample Repository at the Wellcome Trust Sanger Institute. Screening for an AT-rich tolerant enzyme Various commercial enzymes were used to perform a conventional PCR (50 µl total volume) targeted at a 1217 bp locus (Pf3D7_01:55900-57116) with a high AT-con- tent (84%) and a 540 bp locus (Pf3D7_11:1294982- 1295521) with a relatively balanced base composition (positive control). Ten ng of P. falciparum 3D7 genomic DNA was used as template for each PCR. Pf_1 and Pf_11_ama1 oligo pairs (see additional file 1, Table S1) were used at a final concentration of 0.3 µM to amplify the high AT-rich and positive control fragments respec- tively. For each enzyme, two sets of PCRs were per- formed, one using reagents and conditions provided by the manufacturer (standard conditions) and the other deviating only by inclusion of 60 mM TMAC in the reac- tion mixture (optimized conditions). Each set of amplifi- cation condition was repeated at least twice to confirm the finding. See additional file 1, Table S4 for enzymes used and their catalogue numbers. Illumina sequencing libraries Genomic DNA (0.05 - 1 μg in 75 µl TE buffer) from P. falciparum 3D7 or the clinical isolate was sheared for 3 min using a Covaris S2 (Covaris Inc., Woburn, MA).