Sex determination is of utmost concern in the forensic community for developing the biological profile of an individual. Current molecular techniques are successful with high molecular weight DNA, but are inadequate for low quality or quantity DNA samples. In this study, a more sensitive pyrosequencing assay was developed for sexing challenging samples, such as those originating from aged bone or hair shafts. DYZ1, a high copy repetitive element on the Y chromosome, was multiplexed with Ya5 Alu repetitive element as a human and female control. Amplification with biotinylated primers allowed for solid-phase PCR product preparation and pyrosequencing of all DNAs. This assay was developed with high molecular weight male and female control DNAs and resulted in distinct pyrograms for both sexes. This sexing assay was then tested on several suboptimal sources of DNA: fingerprints deposited on glass, shed cells on worn T-shirts, hair shaft extracts, and ancient skeletal remains. Pyrosequencing sexing results were compared to previous sexing results from standard amelogenin, real-time PCR amelogenin, and/or real-time Alu/DYZ1 testing approaches. Of the 216 extracts tested, 59.5% generated a correct sex result using the Alu/DYZ1 assay. If samples that did not yield sequencing results were excluded from the data set, 80.5% of all samples were correctly sexed. Ultimately, the pyrosequencing Alu/DYZ1 sexing assay was more sensitive and accurate than the amelogenin sexing methods.
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