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; http://sourceforge.net/projects/mrsfast/; in the public domain). These conserved sequences were obtained by aligning 242 human genes listed in the Retinal Information Network (https://sph.uth.edu/Retnet/disease.htm; in the public domain) (RetNet) to the sheep genome (v3.1) using the UCSC genome browser and the Galaxy Web-based platform (https://usegalaxy.org/; in the public domain). The obtained BAM-formatted files were analyzed using Genome Analysis Toolkit or GATK (GenomeAnalysisTK-2.7-2; https://www.broadinstitute.org/gatk/; 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 (http://provean.jcvi.org/seq_submit.php; 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