Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 Page 11 of 12 We counted the number of bases in the genome with reads as a percentage of the total mapped reads for each zero coverage (Cov = 0) and less than 5× coverage data set. Mean values are shown (see additional file 1, (Cov<5×). We used SAMtools to generate coverage Table S2). plots and bash/awk scripts for coverage counting. Toler- All datasets used in this study have been deposited in ance to regions of extreme base composition was ana- the ENA read archive under accession number lyzed after normalizing data sets by overall average [ERP000832]. coverage depth. Coverage distribution on selected regions (on chromosome 11) are presented as a boxplot generated in R [15]http://www.r-project.org/. Fidelity Most polymerases introduce errors during base incor- poration in regions of low complexity. To assess these forced errors (or template-dependent errors), we com- pared variant calls by each amplification method at sites of low complexity (mainly TATA repeats). We also assessed the ability of each amplification condition to find a set of known consensus errors in the reference, and used data obtained to score true and false positives. The known consensus errors in the reference genome were previously identified and published [16]. We used the iCORN program [16] to conservatively map reads to the reference genome. iCORN iteratively maps reads and compares them with perfectly mapping reads. PCR-free variant calls were included as a quality control. A mis- match or indel was called with mpileup (a function in SAMtools) at a quality ≥40 from the vcf file. We differ- entiate between a true positive mismatch or indel (if the call agrees with iCORN and PCR-free), and a false posi- tive, (if the call disagrees with iCORN and PCR-free) at that position. Using the total number of errors/variants counted per sample, we generated a normalized error score for each enzyme or library-preparation method. Rank We ranked the results by assigning a score of 1 to the best and a score of 0 to the worst. For each amplifica- tion condition, we calculated the average score value obtained in both coverage and fidelity metrics. Condi- tions with the highest average score was ranked highest while that with the least assigned the lowest rank (see Table 2). Chimeric and duplicate reads analysis We used the BWA paired-end alignment algorithm to generate mapped data sets. Picard software http://picard. sourceforge.net/command-line-overview.shtml was used to identify/mark duplicates from the mapped data set. Chimeric reads were identified from the alignment results using the “bwasw” option with single-end reads. Each replicate data set was analyzed independently to determine the number of chimeric or duplicate reads. We calculated the number of chimeric or duplicate Additional material Additional file 1: Figure S1. Bioanalyzer quantification and analysis of libraries. Aliquots of the PE-adapter-ligated library (shown) was amplified using enzymes/conditions indicated. After purification with Agencourt Ampure XP beads, library products were analyzed using Bioanlyzer. Bioanalyzer traces show various yields obtained by different conditions and amplification methods used. T7 and PCR-free produced the lowest yield. Phusion and AccuPrime amplifications were undetectable in the presence of TMAC (Tetramethylammonium chloride). Additional file 1, Figure S2. Artemis screen view of coverage. Effect of GC content on coverage for a PCR-free library and four other amplified libraries under test with P. falciparum 3D7 chromosome 1. A) Coverage over the entire chromosome. B) Coverage over high AT-content locus. Kapa HiFi and Platinum pfx libraries shown were amplified in the presence of TMAC. (See Figure 3C for coverage of the GC rich telomere). Additional file 1, Figure S3. Box plots showing coverage analysis of P. falciparum chromosome 11. The effect of TMAC on Kapa HiFi and Kapa2G library amplification. A) Coverage plot for each library on the entire chromosome. Subplots B, C, and D shows coverage of sub-regions of the P. falciparum 3D7 chromosome 11. B) Shows base coverage distribution for each library over gene Pf11_0074 and its neighboring introns. C) Coverage at positions 259985-260864 (extreme AT-region). D) Coverage at positions 29092-30361 (VAR gene and introns). Additional file 1, Table S1. Oligonucleotides used in this study. *Phosphorothioate linkages protect the overhanging thymidine from exonuclease activity. Additional file 1, Table S2. Chimera and duplicate analysis. The average number of chimeric or duplicate reads identified from mapping results is shown for each data set. Percentage of mapped data that were found to be chimeric or duplicated in each data set is shown. Mapped reads were normalized to 21× genome coverage. The two isothermal amplifications generated the highest number of chimeric and duplicate reads. Mapped reads were lower for the duplicate analysis than the chimera analysis because of the different alignment methods used. Additional file 1, Table S3. Ranking analysis. Raw ranking data for P. falciparum 3D7 genome coverage and accuracy scores. Mapped reads were normalized to 21× genome coverage. The number of True positive mismatches, False positive mismatches, Deletion and Insertions identified in each library data sets were used to generate a score before combining all scores to generate an overall mismatch rank. Cov, coverage; MM, mismatch; TP, true positive; FP, false positive; DEL, deletion; IN, insertion. Additional file 1, Table S4. Commercial DNA polymerases used and their catalogue number. Abbreviations TMAC: Tetramethyammonium chloride; PCR: Polymerase Chain Reaction; NGS: Next-Generation Sequencing; PE: Paired-end; qPCR: quantitative PCR; RPA: Recombinase Polymerase Amplification; LADS: Linear amplification for deep sequencing; SNP: Single Nucleotide Polymorphism. Acknowledgements This work was supported by the Wellcome Trust [grant number 079355/Z/ 06/Z]. Author details 1Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK. 2Oxford Nanopore Technologies, Edmund Cartwright House, 4 Robert Robinson Avenue, Oxford OX4 4GA, UK.