Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 Page 6 of 12 (i) h t p e d e g a r e v o c d e z i l a m r o N (ii) h t p e d e g a r e v o c d e z i l a m r o N Figure 4 Box plots showing coverage analysis of P. falciparum chromosome 11. (i) P. falciparum 3D7; mapped reads normalized to 21× genome coverage (1 normalized depth represents 21×). (ii) Clinical isolate PK0076; mapped reads normalized to 11× genome coverage (1 normalized depth represents 11×). Subplots B, C and D in both i & ii show coverage of sub-regions of the P. falciparum 3D7 chromosome 11. A) Coverage depth variability plotted for each library on the entire chromosome. B) Distribution of base coverage depth for each library over gene Pf11_0074 and its neighboring introns. C) Distribution of base coverage depth at positions 259985-260864 (extreme AT-region). D) Distribution of base coverage depth at positions 29092-30361 (VAR gene and introns). Top and bottom sides of a box plot represent 75th and 25th percentile of base coverage-depth distribution respectively. The middle line represents 50th percentile. A narrow box indicates less variation in coverage depth across that locus and vice versa. Kapa HiFi, Kapa2G and Platinum pfx enzymes were used in the presence of TMAC. All P. falciparum 3D7and most clinical isolate libraries were prepared in duplicate and each replicate data plotted independently as shown. consistency in coverage depth than Phusion, Kapa HiFi produced the best coverage at the AT-rich regions with evenness almost matching that of PCR-free library. At this AT-rich locus in 3D7 (Figure 4i-C), 75th and 25th percen- tiles of base coverage depths by both Kapa HiFi libraries produced ~18× and 13× coverage respectively. On the other hand 75th and 25th percentile of base coverage depth by both Phusion libraries produced ~13× and 3× coverage respectively. The wider gap in coverage depth distribution by Phusion-amplified library indicates uneven and poor coverage of AT-rich regions. For all conditions, coverage of positions 29092-30361 (VAR gene and introns) was generally very low and inconsistent in the clinical isolate (Figure 4(ii) D) compared to 3D7 (Figure 4 (i) D), perhaps due to the antigenic variation phenomenon associated with these genes. Overall, we show that our optimized conditions improve the quality of coverage in both clinical and non-clinical samples. Isothermal exponential amplification of Illumina genomic libraries using RPA technology High extension temperatures in conventional PCR proce- dures have been associated with bias and other negative effects on high AT content libraries. We therefore assessed the suitability of an isothermal exponential amplification method provided by the recombinase poly- merase amplification (RPA) technology [11]. RPA uses recombinase enzymes which bind oligonucleotide pri- mers (single-stranded DNA) to form nucleoprotein fila- ments that stimulate searching for homologous sequences in a duplex-DNA template. The primer- recombinase complex is able to attach to the homologous DNA duplex and facilitate strand exchange, eliminating the need to denature the template as in PCR. Disassem- bly of the recombinase allows strand-displacing DNA polymerase to begin primer extension from the 3’ end. Two opposing oligonucleotide primers initiate an iso- thermal exponential amplification through automatic cyclic repetition of this process. We used RPA technology (TwistDx) with Illumina PE primers to amplify adapter- ligated libraries before sequencing. We obtained a rela- tively high yield of RPA-amplified library compared to T7 libraries (see additional file 1, Figure S1). Analysis of sequence data obtained from RPA-amplified libraries showed reduced bias towards GC rich templates com- pared to standard Illumina libraries for both P. falci- parum 3D7 and the clinical isolate (Figure 3A & 3B). Coverage of extreme AT base-composition loci was