Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 those caused by AT-rich parts of the genome [4,5]. Since completion of the malaria genome sequencing and the subsequent development of massively parallel sequencing technology, there has been increased effort in using genetic information obtained through re- sequencing initiatives to control and ultimately elimi- nate malaria (Manske et al, submitted). The Wellcome Trust Sanger Institute is currently using Illumina NGS technologies to re-sequence thousands of clinical iso- lates from various malaria endemic regions, mainly sourced from Africa and Asia. Re-sequencing of these samples has faced two major challenges: low mass of total parasite DNA and extremely high AT-base com- position of the genome. These problems pose major technical challenges predominantly in the library pre- paration stage of the NGS pipeline and also in subse- quent data analysis. Due to extreme AT bias, standard Illumina library preparation procedures produce libraries with poor representation that are generally low in complexity compared to the genomic DNA from which they were derived. Polymerase chain reaction (PCR) amplification conditions as currently used in the standard library preparation procedures, have been shown to introduce biases in sequence coverage towards DNA regions with balanced base composition[5,6]. Furthermore, genomic regions with high AT or very high GC content generally result in little or no amplifi- cation at all. These PCR-introduced artifacts have been shown to cause misleading or inaccurate conclusions in the analysis of genome-variation data [4]. Attempts have been made to overcome coverage pro- blems associated with PCR amplification by omitting the PCR step in library preparation[6]. Although the PCR-free approach has improved read distribution and produced more even genome coverage, it requires large amounts of starting DNA material, which is difficult to obtain especially from clinical isolates. The low mass of DNA in clinical iso- lates demands some form of amplification to generate suffi- cient quantities for an Illumina library preparation. We have therefore investigated various amplification possibili- ties and developed library-preparation procedures that are optimal for extremely AT-rich genomes using the Illumina sequencing platform. Here we report alternative library-amplification proce- dures optimized for AT-rich templates and suitable for low mass DNA starting material especially from malaria clinical isolates. We have tested various library amplifica- tion methods including linear and isothermal amplification strategies. We have also explored various PCR amplifica- tion conditions by testing different polymerases, and their tolerance to AT-rich templates, in the absence or presence of tetramethylammonium chloride (TMAC), a PCR addi- tive that increases thermostability of AT base pairs [7]. Page 2 of 12 Results Conventional PCR amplification of an AT-rich locus In order to identify polymerases and PCR conditions that are efficient and tolerant to high AT content templates, we performed conventional PCR using commercial enzymes. The PCR was targeted at a 1217 bp locus (Pf3D7_01:55900-57116) with a high AT-content (84%) and a 540 bp locus (Pf3D7_11:1294982-1295521) with a relatively balanced base composition (positive control) from the P. falciparum 3D7 genome http://www.plas- modb.org[8]. Pf_1 and Pf_11_ama1 oligo pairs (see addi- tional file 1, Table S1) were used to amplify the high AT- rich and positive control fragments respectively. For each enzyme, two sets of PCRs were performed, one using reagents and conditions provided by the manufacturer (standard conditions) and the other deviating only by inclusion of 60 mM TMAC in the reaction mixture (opti- mized conditions). TMAC is a DNA-binding reagent that has been shown to increase the melting temperature of AT base pairs, thereby improving thermostability of the AT-rich regions during PCR amplification. Although in the presence of 3M TMAC, the thermostability of AT base pairs is equal to GC base pairs, this concentration is also inhibitory to polymerase activity. It has been shown that low concentrations of TMAC (60 mM) improve AT base pair stability and result in increased specificity and yield of PCR products [7]. As shown in Figure 1, most enzymes tested were able to amplify the relatively balanced base composition fragment in the absence of TMAC additive, although with different efficiencies. The presence of TMAC inhibited the activity of most enzymes except for Kapa HiFi, Kapa2G Robust and Plati- num pfx, where the additive had a positive effect in amplifying the base-balanced region. At the extreme AT- rich region, nearly all of the enzymes tested were unable to produce detectable amplification either in the presence or absence of TMAC additive. AccuPrime Taq HiFi and Phusion enzymes produced low but detectable amplifica- tion of the test region only in the absence of TMAC. However, Kapa HiFi and Kapa2G Robust were the only enzymes that showed efficient amplification of the AT- rich locus in the presence of TMAC additive. Interest- ingly, the two Kapa enzymes did not produce detectable amplification of this locus in the absence of TMAC, which demonstrates the effect of TMAC on an AT-rich template. PCR amplification of the two test loci were per- formed at least twice for each enzyme tested and in each case the same results were obtained. Enzymes and PCR conditions that showed some level of amplification of the AT-rich locus were used to amplify genomic libraries for Illumina sequencing. We analyzed the sequence data to assess suitability of each polymerase and conditions for amplification of AT-rich libraries.