Probabilistic genotyping replicate analysis of FaSTR clustered single sperm aSTR haplotypes reconstitutes probative diploid DNA genotypes from complex semen mixtures.

Analysis of individual single sperm can assist in mixture deconvolution when there are multiple perpetrators to a sexual assault or instances in which limited sperm are available for analysis. This not only allows for reconstitution of the sperm donor's genotype but also allows for microscopic confirmation that the cell typed was a sperm cell. Single sperm analysis can be challenging due to sperm's low DNA template amount (~3.3 pg) and haploid nature, allowing for only half the donor's genotype to be detected in a single sperm cell. This can make clustering single sperm by donor difficult, as each sperm sample results in a different multilocus haplotype; although, some degree of allele sharing is expected between single sperm from the same donor. In the present work, FaSTR™ was validated for single sperm analysis. The database matching and sample-to-sample matching features of FaSTR™ were used to develop a clustering method to group autosomal short tandem repeat (aSTR) genotyped single sperm by donor. Once clustered, probabilistic genotyping replicate analysis (STRmix™) was conducted in a proof-of-concept investigative manner, resulting in highly probative single source 99% component diploid DNA genotypes which could be used for database (e.g., known offender database) searching when a reference is unavailable. This approach was applied to two different 2-, 3-, and 4-person semen mixtures (total of 6 mixtures) resulting in the recovery of highly probative single source genotypes.