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Quality assessment, validation and analysis of RNA-Seq data.

(A) Bar diagram illustrating the mapping statistics of the indicated samples. “Multiple loci” indicates reads that map to up to 10 loci, “too many loci” refers to reads that map to more than 10 loci. (B) Quantitative measurements of mRNA expression for cypa3, rbp4, si:dkey-18a10.3, apoa1, and arntl1a (bmal1a) by RT-qPCR (x-axis) highly correlate with the corresponding RNA-Seq data (y-axis). The Spearman’s correlation coefficient for the values determined by the two techniques is 0.8697 (p-value <0.0001). (C) The mutant specific alterations in gene expression as determined by the RNA-Seq study for zgc:158291, lhx4, and apoa1 are detectable in 24 hpf embryos using whole-mount in situ hybridization. zgc:158291 is expressed in cranial ganglia (anterodorsal lateral line ganglia (gAD) and statoacoustic ganglion(gVIII)) across all phenotypes (siblings, rx3 weak, and rx3 strong). The expression domain in the optic fissure (of) is lacking in both blind mutants. lhx4 is highly expressed in wild-type siblings in the pineal (p), the trigeminal ganglion (gV) and the adenohypophyseal placode (ap). The expression of lhx4 is affected in a gradual manner: rx3 weak mutants still exhibit a weak expression in the presumptive adenohypophysis. rx3 strong mutants lack the expression domain completely. apoa1 expression in the subpallial telencephalon (sp) shows a broad expansion exclusively in rx3 strong mutants. (D,E) Biological functions enriched in model 11 genes (D) and model 5-6-14-15 genes (E). (F,G) Metabolic pathways enriched among model 11 genes (F) and model 5-6-14-15 genes (G).