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BMC Res Notes
2015 Nov 18;8:691. doi: 10.1186/s13104-015-1485-1.
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RNA-Seq and microarray analysis of the Xenopus inner ear transcriptome discloses orthologous OMIM(®) genes for hereditary disorders of hearing and balance.
Ramírez-Gordillo D
,
Powers TR
,
van Velkinburgh JC
,
Trujillo-Provencio C
,
Schilkey F
,
Serrano EE
.
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Auditory and vestibular disorders are prevalent sensory disabilities caused by genetic and environmental (noise, trauma, chemicals) factors that often damage mechanosensory hair cells of the inner ear. Development of treatments for inner ear disorders of hearing and balance relies on the use of animal models such as fish, amphibians, reptiles, birds, and non-human mammals. Here, we aimed to augment the utility of the genus Xenopus for uncovering genetic mechanisms essential for the maintenance of inner ear structure and function. Using Affymetrix GeneChip(®) X. laevis Genome 2.0 Arrays and Illumina-Solexa sequencing methods, we determined that the transcriptional profile of the Xenopus laevis inner ear comprises hundreds of genes that are orthologous to OMIM(®) genes implicated in deafness and vestibular disorders in humans. Analysis of genes that mapped to both technologies demonstrated that, with our methods, a combination of microarray and RNA-Seq detected expression of more genes than either platform alone. As part of this study we identified candidate scaffold regions of the Xenopus tropicalis genome that can be used to investigate hearing and balance using genetic and informatics procedures that are available through the National Xenopus Resource (NXR), and the open access data repository, Xenbase. The results and approaches presented here expand the viability of Xenopus as an animal model for inner ear research.
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Fig. 1. Experimental strategy for determining the expression of OMIM® orthologues for deafness and vestibular disorder genes in the Xenopus inner ear transcriptome with Illumina-Solexa (RNA-Seq) and Affymetrix microarray methods. A comprehensive list of OMIM® genes for deafness and vestibular genes was manually curated and used to map OMIM® sequences to the Xl-PSIDs on the Affymetrix GeneChip
®
X. laevis Genome Array, and to the JGI Xenopus reference genome scaffolds. Xenopus inner ear RNA was used in microarray hybridization reactions with the Affymetrix GeneChip
®. The hybridization data were analyzed to retrieve intensity values from target Xl-PSIDs that had met alignment criteria for OMIM® orthologues for deafness and vestibular disorder genes. The Alpheus® program was implemented to map inner ear RNA-Seq reads to the JGI Xenopus reference genome scaffolds and the RNA-Seq alignment data were analyzed to retrieve target scaffold regions that had met alignment criteria for OMIM® orthologues for deafness and vestibular disorder genes. As part of the analysis, the data were separated into three OMIM® phenotype categories: deafness only; vestibular disorder only; and both deafness and vestibular disorder. When expression criteria were applied to both datasets, RNA-Seq methods detected expression of more OMIM® orthologues for deafness and vestibular disorder genes in the Xenopus inner ear (241) than were detected by microarray methods (126)
Fig. 2. Comparison of Illumina-Solexa (RNA-Seq) and Affymetrix microarray methods for detection of Xenopus orthologues for OMIM® deafness and vestibular disorder genes. Bar graphs compare detection data for 131 deafness only OMIM® genes (a), 28 vestibular disorder only OMIM® genes (b), 31 OMIM® genes that are associated with both vestibular and deafness phenotypes (c) and all 190 OMIM® genes (d) that met alignment criteria for both microarray and RNA-Seq analysis. Both technologies met expression criteria for 108 (57 %) of the 190 OMIM® genes. RNA-Seq met expression criteria for 48 (25 %) additional OMIM® genes not detected by microarray. Eleven (6 %) genes were detected by microarray methods only. Expression criteria were not met with either technology for 23 (12 %) of the 190 Xenopus OMIM® orthologues that met alignment criteria
Fig. 3. Correlation plot of Affymetrix microarray and Illumina-Solexa (RNA-Seq) measurements of OMIM® gene expression. Data (log2) from 108 Xenopus orthologues for OMIM® deafness and vestibular disorder genes that met expression criteria for both technologies are plotted in the figure (X-axis, microarray GCRMA intensity values; Y- axis, RNA-Seq read based intensity value). A modest correlation was detected between RNA-Seq reads and microarray intensity values in this analysis (R = 0.49, R2 = 0.24, P value <0.5)
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