Novel Bioinformatic Analyses of Somatic Cell Contamination in Sperm Samples

To establish contamination profiles, the sperm donors with normal sperm counts were analyzed using an Infinium HumanMethylation450 array. Somatic cell lysis, sperm isolation, DNA extraction, and bisulfite conversion were performed as described by Aston et al. The bisulfite converted sperm DNA was hybridized to Illumina Infinium HumanMethylation450K microarrays at the University of Utah and run as recommended by the manufacturer (Bibikova et al. 2011). Unpaired blood samples were extracted using Qiagen's DNeasy Blood and Tissue kit and bisulfite converted using Zymo's EZ DNA Methylation kit. All procedures were performed according to the instructions of the manufacturer. Four permutations were run on each sample, including pure blood, half blood and half sperm by DNA concentration, half blood and half sperm by cell count, and pure sperm (n = 16). Concentration was normalized using a spectrophotometer. A Makler cell counting chamber was used to count white blood cells and sperm, which were then normalized in a 1:1 ratio. Bisulphite converted DNA from the 16 samples were hybridised to the Illumina Infinium450 Human Methylation Beadchip