SMAD4-associated Myhre syndrome mutations are under positive selection in the male germline

Samples underwent multiple rounds of digestion with the restriction enzyme NsiI followed by PCR amplification. Libraries were then prepared and ultra-deep next-generation sequencing performed on an Illumina MiSeq platform with 2x151 paired-end reads. For the titration assay, one human blood sample spiked in with genomic DNA from two different Myhre syndrome patients at known concentrations was used. For the reproducibility assay, two or four human sperm samples from four different individuals were assessed. For the final assay, 57 human sperm samples and five human blood samples were utilised.