Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 A ) % ( s d a re 4 d e p p a M 8 6 2 0 10 C ThePoCrRe-tfirceael Kapa HiFi AcPcKluaaPtpirnaium2mGe RPA T7 Phusion B 8 6 ) % ( s d a re 4 d e p p a M 2 0 Page 5 of 12 ThePoCrRe-tfirceael Kapa HiFi AcPcKluaaPtpirnaium2mGe RPA T7 Phusion 20 30 GC content (%) 40 50 60 10 20 30 GC content (%) 40 50 60 Figure 3 GC profile analysis of sequenced data. The GC content distribution for different library preparation methods are shown alongside theoretical data for comparison. A) GC content analysis on libraries prepared from P. falciparum 3D7 with mapped reads normalized to 21× genome coverage. B) GC content of libraries prepared from a clinical isolate (PK0076) with mapped reads normalized to 11× genome coverage. Libraries with GC content above 19.4% (the GC content of the P. falciparum 3D7 reference genome) indicate amplification bias towards templates with neutral GC composition. C) Artemis [9,10] screen view of coverage (mapped reads normalized to 21× genome coverage) for a PCR-free library and four other libraries under test on P. falciparum 3D7 chromosome 1 (zoomed in to show coverage on the GC rich telomere). Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. See additional file 1, Figure S2 A & B for coverage on the entire chromosome 1 and AT-rich locus. free library in coverage of either extremes of base composition. We also assessed coverage across chromosome 11 and then focused on specific regions of balanced GC content, high AT content and variable base compositions. A box plot showing variation in depth coverage for both P. falciparum strain 3D7 and the clinical isolate is shown on Figure 4i & ii respectively. In these analyses, we observed relatively even coverage depth across the base- balanced region (panel B of Figure 4i & ii) by all the enzymes and conditions tested (this is indicated by the narrow gap between the 25th and 75th percentile of base coverage distribution). However, large variations in cover- age depths were observed across the AT-rich and the vari- able base composition loci (Figure 4i & ii panels C and D respectively). Although most enzymes showed more Table 1 Average GC content Sample Library 3D7 Av. %GC content PK0076 Av. %GC content PCR-free 19.50 21.92 Kapa HiFi 20.35 19.79 Kapa2G 21.44 21.07 AccuPrime 21.93 21.37 Platinum 21.47 21.91 RPA 20.75 19.66 T7 22.63 20.95 Phusion 23.80 22.87 Calculated values of average GC content corresponding to each library preparation. Deviation from the 19.4% (value of the unamplified genome) indicates the effect of amplification bias. See Figure 3 A & B for a global graphical presentation of the GC content distribution. 3D7, P. falciparum strain 3D7; PK0076, P. falciparum clinical isolate.