Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 continue in an incubator block at an isothermal tempera- ture of 37°C for 40 min. Amplification products were puri- fied with 1× Agencourt Ampure XP beads (Beckman Coulter) and eluted in EB buffer (Qiagen). Illumina linear library amplification using T7 RNA polymerase Linear amplification for deep sequencing (LADS) [12] was used to amplify end-repaired and A-tailed genomic DNA library fragments. Starting with 200 ng of genomic DNA, ~30 ng of A-tailed library fragments were ligated to Adap- ter-A and Adapter-B, previously prepared by annealing adapter-A and adapter-B forward and reverse oligos (see additional file 1, Table S1) respectively. Adapter-ligated libraries were cleaned twice with 1× Agencourt Ampure XP to remove non-ligated adapters and adapter dimers. Alternatively, adapter-ligated library was electrophoresed in a 2% agarose gel alongside a DNA ladder and fragment bands of 250 to 600 bp were cut and purified at room temperature using the Qiaquick gel extraction kit (Qia- gen). The purified adapter-ligated library (20 ng) was used to perform in vitro transcription at 37°C overnight with reagents from the MEGAscript T7 kit (Ambion). The pro- tocol described in [12] was followed to generate T7-ampli- fied libraries ready for Illumina cluster generation and sequencing. The procedure was repeated twice, each time starting from same genomic DNA, to generate technical replicates PCR-free libraries Genomic DNA (1-2 μg) was sheared, end-repaired and A-tailed as described under “Illumina sequencing libraries”. Pre-annealed paired-end Illumina No-PCR adapters were ligated using the same ligation procedure described under “Illumina sequencing libraries”. Result- ing adapter-ligated fragments were loaded alongside a low molecular weight ladder (NEB) on a TBE 2% agarose gel. Fragment bands of 350 to 500 bp were cut and puri- fied using a Qiaquick gel extraction kit. Gel purification was performed at room temperate and DNA eluted in EB buffer. Library quantification by qPCR Pre-PCR libraries were quantified by qPCR using a pre- viously quantified and sequenced library in dilutions containing 100 ng, 10 ng and 1 ng as standard. The qPCR reaction (25 μl) contained 12.5 μl of 2× Sybr Green PCR Master Mix (Applied Biosystems), 0.75 μl of 10 μM Pre-PCR qPCR top and bottom primers (see additional file 1, Table S1), 2.5 μl of library sample (pre- diluted 1000 fold) and 8.5 μl of PCR-quality water. The reaction was set in triplicates and run on a StepOne™ Real-Time PCR System (Applied Biosystems) with a thermocycling protocol of 10 min at 95°C for the initial Page 10 of 12 denaturation followed by 40 cycles of [30 s at 95°C and 1 min at 60°C]. Prior to cluster generation, amplified or no-PCR libraries were quantified by qPCR using the SYBR fast Illumina library quantification kit (KAPA Bio- systems). The qPCR was done following instructions provided by the kit manufacturer and run on the Ste- pOne™ Real-Time PCR System (Applied Biosystems). The thermocycling protocol was 5 min at 95°C for the initial denaturation followed by 35 cycles of [30 s at 95° C and 45 s at 60°C]. Data processing Replicate data sets were analyzed independently by map- ping sequence reads to the P. falciparum 3D7 reference genome V2.1.5 ftp://ftp.sanger.ac.uk/pub/pathogens/ Plasmodium/falciparum/3D7/3D7.version2.1.5/ using BWA [13]. SAMtools [14] was used to generate cover- age data from the BWA pileup mapping output. To compare the quality of sequence data from different library preparation methods, we developed four analysis metrics. Evenness of coverage depth metrics - compar- ing the overall representation and depth across the entire genome; GC content analysis - to detect base composition biases in amplification through comparison with a theoretical GC content derived from shredded reads of the reference genome [6]; Tolerance metrics - to assess representation of extreme base-composition loci, focusing on selected genomic regions; and Fidelity metrics - assessing enzyme-dependent errors. Genome coverage Mapped reads for each replicate data sets were normal- ized by average genome coverage to 11× and 21× for clinical and non-clinical samples respectively, and then pooled or analyzed independently depending on analysis metrics used. For the clinical samples, reads originating from host contamination were filtered out (by aligning all reads to P. falciparum 3D7 reference genome) prior to analysis, hence the relatively low average genome coverage depth for these samples. Evaluation of coverage depth and evenness was based on cumulative distribu- tions over a normalized overall average depth. A mea- surement of low-coverage index lci (d) is defined as the integration of the cumulative distribution C(x) from 0 to d giving an overall assessment of the coverage at the low end of distribution: lci(d) = 0 d C(x)dx The value lci (0.5) that gives a measurement of the coverage below half of the average depth in the distribu- tion was used to compare evenness of coverage for each data set.