Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 Page 3 of 12 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Figure 1 Screening for tolerance to an AT-rich template using conventional PCR amplification. Top panel: PCR amplification of a 540 bp locus (Pf3D7_11:1294982-1295521) with a relatively balanced (70% AT) base composition (positive control) in the presence or absence of TMAC. Bottom panel: PCR amplification of a 1217 bp locus (Pf3D7_01:55900-57116) with extreme AT content (84%) in the presence or absence of TMAC. M, 100 bp DNA ladder (NEB); (1) PWO master; (2) PWO master + TMAC; (3) PfuULTRA; (4) PfuULTRA + TMAC; (5) Kapa HiFi; (6) Kapa HiFi + TMAC; (7) AccuPrime Taq HiFi; (8) AccuPrime Taq HiFi + TMAC; (9) AccuPrime pfx SuperMix; (10) Phusion; (11) Phusion +TMAC; (12) Platinum HiFi; (13) Platinum HiFi + TMAC; (14) Platinum pfx; (15) Platinum pfx + TMAC, (16) Ex Taq; (17) Ex Taq + TMAC; (18) Kapa2G Robust; (19) Kapa2G Robust + TMAC. PCR amplification of Illumina genomic libraries Although Phusion polymerase is the standard enzyme for Illumina sequencing library amplification, it produces libraries with poor representation of loci with extreme base composition particularly the AT-rich regions [4]. In our screening above, many enzymes failed to show effi- cient amplification of the test locus and were clearly intolerant to AT-rich templates. We evaluated the effi- ciency of genomic library amplification using poly- merases that showed some tolerance to the AT-rich template. We tested Phusion, AccuPrime Taq HiFi, Plati- num pfx, Kapa2G Robust and Kapa HiFi. We used these enzymes to amplify PE adapter-ligated genomic libraries made from malaria parasite clinical isolate and the refer- ence strain P. falciparum 3D7. In the presence of TMAC, AccuPrime Taq HiFi and Phusion produced undetectable amplifications that were not processed further. However, alternative amplification conditions produced high ampli- fication yields (see additional file 1, Figure S1), that were sequenced and analyzed. All sequence data sets generated were normalized to average coverage before analysis. Two data sets were generated in parallel: One from a lab- cultured parasite (P. falciparum strain 3D7) genomic DNA; and the other one from P. falciparum clinical iso- late containing ~53% host DNA contamination. Sequence data from the clinical isolate were filtered to remove host contamination before analysis resulting in lower coverage depths compared to those from 3D7. To analyze and compare the data generated under optimized conditions against standard procedures, we developed analysis metrics that assess four aspects of data quality: Evenness of coverage depth metrics was used to analyze the distribution of coverage depth for each library across the entire genome and plot results of multiple data sets in a single graph for comparative analysis; GC content analysis was used to compute the average GC content for each library and assess base-composition biases; toler- ance metrics was used to assess representation of extreme base-composition loci, focusing on selected regions; and fidelity metrics was used to assess enzyme- dependent amplification errors. Using these metrics, we show that libraries prepared under standard conditions (using Phusion polymerase) produced less even coverage compared to the optimized conditions in both 3D7 and the clinical isolate. As shown in Figure 2A & 2B, up to 30% of the genome was covered at depths of ≤10× for 3D7 and ≤5× for Clinical isolate in Phusion-amplified libraries. In contrast libraries amplified under optimized conditions had most of the genome cov- ered at high depths and only <10% of the genome covered at depths ≤10× and ≤5× for 3D7 and clinical isolate respectively. Libraries prepared under standard conditions also showed strong bias towards the GC-rich templates as shown by a shift in average GC content from ~19.4% (un- amplified reference genome) towards GC neutral base composition (Figure 3A & 3B). Table 1 shows the average GC content for each amplification condition tested. To illustrate this further, we used artemis [9,10] to view mapped reads on chromosome 1 and assess coverage depth distribution with respect to base composition as depicted by a GC content plot (see Figure 3C and addi- tional file 1, Figure S2 B). We show that under standard conditions, high GC content regions across chromosome 1 including telomeres were over-amplified whereas regions of high AT content were under-amplified. On the other hand Kapa HiFi amplification was close to that of a PCR-