MOESM1 of Bead-based assays to simultaneously detect multiple human inherited blood disorders associated with malaria

Additional file 1: Table S1. Optimisation flow chart: A. Genomic PCR, B. ASPE (allele specific primer extension) and C. Hybridisation of ASPE reactions to microspheres. Optimal conditions in black. Table S2. Cut-off MFI values for each marker. The mean plus two standard deviations (Mean + 2SD) of the background MFI signal of each marker was chosen as a cut-off value. An MFI signal greater than the cut-off scored a positive result. Table S3. Examples of how the net median fluorescence intensity (MFI) is recorded for each allele and the percentage wild type MFI (WT %) is calculated. A. For G6PD (G202A and A376G) in males (males only have a single copy of the G6PD gene), above 75% wild type MFI signifies that the sample is wild type and below 25% wild type MFI signifies that the gene carries a mutation. In males, if the percentage wild type MFI is between 25%-75%, the genotyping needs to be repeated. In HBB (HbC and HbS), above 75% wild type MFI signifies homozygous wild type and below 25% wild type MFI signifies a mutation on both alleles. A percentage wild type MFI and a percentage mutated MFI in between 25% and 75% signifies that the sample is heterozygous and contains both a wild type and mutated allele. The G6PD genotypes are as follows: A- samples carry both the 202A and 376G mutations and the A+ samples carry the 202G wild type allele and the 376G mutation. B. The HBB genotypes can be either homozygous HbAA, HbCC or HbSS. Heterozygous genotypes contain a combination of genotyping calls, for example, HbA/C samples are heterozygous samples with a wild type allele (HbA) and a mutated HbC allele. This sample does not contain the HbS mutation on either of its two alleles. An HbA/C/S genotype is heterozygous for the both HbC and HbS alleles. Samples with net MFI values below the mean MFI plus two standard deviations (Mean + 2SD) for that marker are highlighted with a * (see Table S2). These samples were excluded from further analysis. Table S4. G6PD and HBB genotyping on samples from female participants. Six samples from females were analysed to show that the assay is working for homozygous and heterozygous women. Table S5. Pricing and time. Figure S1. Example of comparison of G6PD activity in normal (left) and deficient (right) individuals. The grey-shaded peaks correspond to samples and the blue-outlined peaks correspond to untreated negative controls. The log-10 fluorescence in the FL1 channel (533 ± 30 nm) is measured (x-axis). The SE Dmax % positive and the Overton % positive detect the % of positive cells against the control. The SE Dmax additionally accounts for outliers. Figure S2. Flow cytometry results by G6PD genotype. The percentage of G6PD positive cells was plotted by G6PD genotype generated by magnetic bead-based multiplex assay; wild type (wt, 202G and 376A), A+ (202G and 276G) and A- (202A and 376G). The % G6PD positive cells were calculated using FlowJo version 10 Super-Enhanced Dmax Subtraction (SED) algorithm.