Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 however inferior to Kapa enzymes (Figure 4i & ii panel C). Furthermore, RPA amplified libraries showed rela- tively high levels of chimeric and duplicate reads (see additional file 1, Table S2). Linear amplification of Illumina genomic libraries using T7 RNA polymerase Exponential amplification of sequencing libraries is thought to be the source of biases and artifacts. To avoid this, we explored a recently described linear amplification for deep sequencing (LADS) protocol [12]. LADS uses T7 RNA polymerase to transcribe DNA fragments bear- ing a T7-promoter sequence at the 5’ end. This is an in-vitro transcription system whereby the T7 RNA poly- merase transcribes a DNA fragment multiple times pro- ducing several RNA transcripts under isothermal conditions and resulting in a linearly amplified template. The resulting RNA material is then converted to cDNA using a reverse transcriptase. We sequenced the T7 poly- merase-amplified libraries and analysed the quality of the data generated using our analysis metrics. The overall yield of the library generated using the LADS protocol was very low compared to those amplified exponentially (see additional file 1, Figure S1). However, coverage of extreme base-composition loci was better than standard Illumina libraries. Libraries produced under optimized PCR conditions were generally superior to the LADS pro- tocol (T7) in coverage of AT-rich loci for both P. falci- parum 3D7 and the clinical isolate (Figure 4 I & ii panel C). Surprisingly, an over-representation of fragments with high GC content was observed in T7-amplified library when viewing sequence reads aligned to the refer- ence genome using artemis [10] (data not shown). T7 polymerase-amplified libraries were also associated with the highest level of duplicate and chimeric reads (see additional file 1, Table S2). Effect of TMAC on Illumina genomic library amplification TMAC has been shown to improve thermostability of AT-base pairs [7]. However, it inhibits the activity of most polymerases. In our analysis, Phusion and Accu- Prime Taq HiFi were inhibited by TMAC and produced no library amplification (see additional file 1, Figure S1). On the other hand Platinum pfx and the Kapa enzymes were tolerant to the concentration of TMAC used in this assay (Figure 1). We analyzed and compared libraries prepared with Kapa enzymes in the presence and absence of TMAC. Our analysis indicates that the overall even- ness in coverage across the genome was significantly improved in the presence of TMAC (see additional file 1, Figure S3). We observed less bias towards GC rich sequences and an overall improved coverage on AT-rich regions. All amplification data shown for Kapa HiFi, Kapa2G and Platinum pfx in Figures 2, 3 and 4 were Page 7 of 12 performed in the presence of TMAC. The positive effect of TMAC was observed in both P. falciparum 3D7 and the clinical isolate (containing ~53% host contamination). Fidelity and coverage ranking We analyzed the accuracy of each enzyme in terms of base incorporation by counting mismatches and indels at regions of low complexity (TATA repeats). At these regions of low complexity, RPA generated the least num- ber of errors (scoring best) whereas T7 polymerase had the highest (Table 2). Kapa HiFi showed a relatively low mismatch score which was largely due to high number of insertions observed at TATA repeat regions where most of the enzymes tested showed little or no coverage. Enzymes were also scored on evenness of coverage depth by counting the number of regions with zero and less than 5× base coverage. Based on these criteria, Phusion had the highest number of regions with zero and <5× coverage, thereby scoring poorest. Kapa HiFi had the best coverage score after PCR-free. Finally, we calculated the overall fidelity and evenness of genome coverage and ranked each amplification condition based on average overall score. As shown in Table 2, libraries amplified by Kapa HiFi came second to PCR-free, whereas Phusion polymerase had the lowest overall score (see additional file 1, Table S3 for raw data used in ranking). Discussion Library amplification is an important step in preparing samples for sequencing with the current NGS platforms, especially when the starting material is severely restricted in quantity. PCR amplification, however, has been shown to be the major source of artifacts and base-composition bias in library fragments. Although alternative library- preparation approaches that omit the PCR amplification Table 2 Ranking of P. falciparum 3D7-amplified and PCR- free libraries. Coverage score MM score Rank ALL 0.99 0.8 0.67 0.59 0.52 0.59 0.22 0.74 0.95 0.58 0.59 0.56 0.6 0.57 0.5 0.43 0.97 0.69 0.63 0.58 0.56 0.58 0.36 0.58 Amplification PCR -Free Kapa HiFi Platinum pfx AccuPrime Taq HiFi RPA Kapa2G Robust Phusion T7 Ranking was based on genome coverage and accuracy. Coverage score: PCR- free and Kapa HiFi had the lowest percentage of genome covered at 0 or <5× thereby scoring the highest. Mismatch (MM) score: Loci of low complexity were used to generate mismatch ranking (see Material and Methods under Fidelity section). The number of True positive mismatches, False positive mismatches, Deletion and Insertions obtained for each library were used to generate a score before combining all scores to generate an overall mismatch rank. Using these criteria, RPA produced the highest score and T7 scored the least. See additional file 1, Table S3 for raw ranking data.