N15 primers

Genomic DNA (gDNA) was extracted from blood samples using Nucleon BACC2 DNA extraction kits [Gen-Probe Life Sciences, Tepnel Research Products and Services, Manchester, UK]. Five nano-grammes of gDNA was whole-genome amplified by primer-extension pre-amplification (PEP) using N15 primers (Sigma, UK) and Biotaq (Bioline, UK) polymerase as previously described by Zhang et al. [17 (link)]. Sixty-eight single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions were selected from the many hundreds of SNPs identified in the literature and on public databases based on the given criteria (1) previous associations with malaria, (2) predicted functional consequences with respect to G6PD enzyme activity (3) estimated minor allele frequency and (4) whether they made a viable Sequenom assay design [18 (link)]. Three gender-typing markers were also included. Assays were performed using the Sequenom® iPLEX platform according to manufacturer’s instruction using diluted PEP DNA (1:10). Genotype calls were made using the Sequenom® Typer v4.03 software [19 (link)].

The blood collected in the EDTA coated tube was used for DNA extraction using QIAGEN QIamp DNA blood mini kit (Cat No: 51106) as per manufacturer’s instructions. Genotyping was done at the laboratory of the Wellcome Trust Centre for Human Genetics, University of Oxford UK. Hundred and seventy SNPs were selected on the basis of a review of reports of associations with malaria data from The MalariaGEN Consortium [25 ]. Genotyping of these selected SNPs (Additional file 1) were carried out by previously described methods [7 (link)]; in summary: five nanograms of gDNA was whole-genome amplified by primer-extension pre-amplification (PEP) using N15 primers (Sigma, UK) and Biotaq (Bioline, UK) polymerase as previously described by Zhang et al. [26 (link)]. Single nucleotide polymorphisms (SNPs) were assayed on the Sequenom^® iPLEX platform according to manufacturer’s instructions using diluted PEP DNA (1:10). Genotype calls were made using the Sequenom^® Typer v4.03 software [25 ].