Computational protocol: Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep

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Protocol publication

[…] DNA extracted from one day-blind and one unaffected Awassi sheep was deep-sequenced using the Illumina (San Diego, CA, USA) HiSeq2000 platform according to the manufacturer's pair-end protocol (ENA accession no. PRJEB12018). Average fragment length was 580 bp, and 100-bp sequence reads were obtained from both ends. Each DNA sample was applied to two lanes yielding ∼11-fold coverage for the sheep genome.Sequence reads were mapped to 5020 conserved-sequence stretches of genes involved in hereditary visual impairment using the Fast Alignment Search Tool suite (mrsFAST-ultra-3.2.0;; in the public domain). These conserved sequences were obtained by aligning 242 human genes listed in the Retinal Information Network (; in the public domain) (RetNet) to the sheep genome (v3.1) using the UCSC genome browser and the Galaxy Web-based platform (; in the public domain). The obtained BAM-formatted files were analyzed using Genome Analysis Toolkit or GATK (GenomeAnalysisTK-2.7-2;; in the public domain) for variant discovery. For each polymorphic site, homozygous variations for the day-blind sheep and heterozygosity or homozygosity for the alternative allele in the normal Awassi sheep were further evaluated by scoring amino acid substitutions using Grantham scores and PROVEAN analysis v1.1 (; in the public domain), which scores the effect of protein-sequence variation on protein function. […]

Pipeline specifications

Software tools mrsFAST, Galaxy, GATK, PROVEAN
Databases RetNet UCSC Genome Browser
Application Genome data visualization
Organisms Ovis aries
Chemicals Amino Acids