Fig. S1. qPCR reactions (singleplex and duplex) and thermal cycling conditions were initially tested using gDNA extracted from D– donors with ccee, Ccee and ccEe phenotypes, and RhD positive donors either hemizygous for RHD with DCcee phenotype or homozygous for RHD with DCcEe phenotype (n = 5). To allow RHD exon 4&10 and RHD exon 5&CCR5 duplexing, RHD exon 4 and 5 TAMRA hydrolysis probes were FAM-labelled (green channel) and RHD exon 10 and CCR5 TAMRA hydrolysis probes were VIC-labelled (yellow channel). a) Green channel analysis of singleplex reactions (RXN), “Exon 4” and “Exon 5” and duplex reactions “Exon 4/Exon 10” and “Exon 5/CCR5”. No Ct values for gDNA samples derived from D– RBC samples with ccee, Ccee and ccEe phenotypes are shown as fluorescence was not above threshold and/or did not exhibit a total change in fluorescence >10% relative to the largest change in any tube. b) Yellow channel analysis of singleplex reactions, “CCR5” and “Exon 10” and duplex reactions “CCR5/Exon 5” and “Exon 10/Exon 4.”

Fig. S2. PCR/agarose gel analysis of RHD(ex8:del/CE) donors using primer set 5&6 and different annealing temperatures. Lane: 1) ladder; 2-7) annealing temperatures 50°C, 52°C, 54°C, 56°C, 58°C and 60°C, respectively. a) RHD(ex8:del/CE) donor #1. b) RHD(ex8:del/CE) donor #2. c) Overexposed gel for RHD(ex8:del/CE) donor #1. d) Overexposed gel for RHD(ex8:del/CE) donor #2. The arrow indicates a non-specific band approximately 400bp in size.


Table S1. The corresponding name, sequence, thermal cycling conditions, RHD position and amplicon size for primer set 1&2-7&8.

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