Abstract
The majority of transcriptome sequencing (RNA-seq) expression studies in plants remain underutilized and inaccessible due to the use of disparate transcriptome references and the lack of skills and resources to analyze and visualize these data. We have developed expVIP, an expression visualization and integration platform, which allows easy analysis of RNA-seq data combined with an intuitive and interactive interface. Users can analyze public and user-specified data sets with minimal bioinformatics knowledge using the expVIP virtual machine. This generates a custom Web browser to visualize, sort, and filter the RNA-seq data and provides outputs for differential gene expression analysis. We demonstrate expVIP's suitability for polyploid crops and evaluate its performance across a range of biologically relevant scenarios. To exemplify its use in crop research, we developed a flexible wheat (Triticum aestivum) expression browser (www.wheat-expression.com) that can be expanded with user-generated data in a local virtual machine environment. The open-access expVIP platform will facilitate the analysis of gene expression data from a wide variety of species by enabling the easy integration, visualization, and comparison of RNA-seq data across experiments.
Highlights
We investigated whether genes commonly used as reference genes in quantitative PCR (qPCR) were stably expressed in our samples
We found that all five genes had low coefficients of variation using qPCR (4.4%–8.4%), suggesting that they are suitable for use as reference genes (Table IV)
We found that the coefficients of variation measured by qPCR were lower than those found by RNA-seq analysis
Summary
Data Preparation ReadsWe downloaded the wheat (Triticum aestivum) gene expression data from the SRA database at NCBI available on August 12, 2015. The source code to prepare and set up the expVIP database and graphical interface are available in Github: https://github.com/homonecloco/expvipweb. The expVIP virtual machine, the data displayed in the Web interface, and the detailed documentation are available on the wiki page https://github.com/homonecloco/ expvip-web/wiki. QPCR was carried out using LightCycler 480 SYBR Green I Master Mix (Roche) with each primer at a final concentration of 0.25 mM and 0.05 mL of cDNA in a 10-mL reaction using 384-well plates. The qPCR program run on the LightCycler 480 (Roche) was as follows: preincubation at 95°C for 5 min; 45 amplification cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s with the final melt-curve step cooling to 60°C and heating to 97°C with five reads per 1°C as the temperature increased. Primer efficiencies were calculated using a serial dilution of cDNA
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