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We developed a 50 SNP-multiplex assay for detection on a MALDI-TOF MS platform based on the SNPs in the 52 SNP-multiplex assay recently developed by the SNP for ID Consortium. After PCR amplification, the products were purified on Qiagen columns and used as templates in one single base extension (SBE) multiplex reaction. Two different strategies were used to design the SBE multiplex: (1) Small 5′-tags (3–8nt) that increased the masses of the SBE primers without changing the annealing temperature; (2) Cleavable primers with one RNA nucleotide which was later cleaved by a mixture of RNases. The SBE primers were extended with biotin labelled ddNTPs and purified on avidin beads ensuring that only the extended SBE primers were isolated and spotted on the MALDI-TOF anchor target. Detection of the 50 extended primers from the SBE reaction was performed in a mass range between 3000 and 10,000 m / z .

The GS FLX pyrosequencing platform using parallel tagged sequencing was tested on 10 Somali individuals for sequencing of the complete mitochondrial genome. The amplicons were sequenced twice with increasing coverage to establish the minimum of coverage needed to produce reliable sequence reads. The genome sequences were compared to previously obtained control regions sequences with Sanger sequencing and 49 SNPs in coding regions of the mitochondrial genome. No discrepancy was found with the three methods except in a poly-C stretch that was estimated to be 16193.1C by sequencing with terminator chemistry and no insert was observed with pyrosequencing. The sequence lengths of both pyrosequencing runs were between 225 and 235 nucleotides. The coverage for the first pyrosequencing run was between 0 and 159 and 10–577 for the second one.