Genetic mapping and sequencing of the prh mutation

Microsatellite markers were tested on at least 30 affected animals to narrow the critical interval using standard methods. Whole genome sequencing was performed with whole brain genomic DNA isolated from an affected mouse by 125 bp paired-end sequencing on the Illumina HiSeq2500 platform. The unique, homozygous, single-nucleotide changes within the minimal genetic interval were selected with the following filtering: (1) within 50 bp of the nearest exon; and (2) not reported as polymorphisms in publicly available sequence variation databases – Sanger Institute Mouse Genome Project (Keane et al., 2011), NCBI dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/), and Celera whole genome shotgun sequence (Mural et al., 2002). Finally, Sanger sequencing was also performed in unaffected siblings to exclude nucleotide changes not associated with the hydrocephalus phenotype. The prh mice were genotyped using the TaqMan Sample-to-SNP Kit (Applied Biosystems) for a single-nucleotide change at chr3:g.33731448A>T (assay ID: AH6R6X7).