ExpressionPlot: a web-based framework for analysis of RNA-Seq and microarray gene expression data

Abstract

RNA-Seq and microarray platforms have emerged as important tools for detecting changes in gene expression and RNA processing in biological samples. We present ExpressionPlot, a software package consisting of a default back end, which prepares raw sequencing or Affymetrix microarray data, and a web-based front end, which offers a biologically centered interface to browse, visualize, and compare different data sets. Download and installation instructions, a user's manual, discussion group, and a prototype are available at http://expressionplot.com/.

Figure 1

The ExpressionPlot home page. The website opens with this screen, giving a list of tools available in ExpressionPlot, and a login box in the top right. The navigation bar on top appears on all pages, giving links to the other tools. The 'manual' link is context-aware: it automatically opens the User's Guide (in another tab) to the page explaining the current tool.

Figure 2

Screen shots of ExpressionPlot quality control tools. (a) read_types tool showing all read types. Numbers of non-aligning (Nonmatch), mulitply-aligning (Mult), unique genome-aligning (Genomic) and unique junction-aligning (Junction) reads are shown for each lane from a mouse tissue transcriptome dataset [3]. Numbers (1/2) indicate different libraries; letters (A/B/C) indicate different lanes of the same library. (b) read_types tool showing matching read types, normalized to 100%. (c) Pairwise correlation heatmap of gene expression profiles generated from each lane. (d) pairdist tool shows ECDF of paired-end distances of 'canonical' reads (same chromosome, different strand, minus strand read downstream of plus strand read). 'Distance' is defined as the genomic distance, in nucleotides, between the aligned positions of the last sequenced bases of the two reads (can be negative if the alignments overlap). The samples have been de-identified (data in Additional file 3). Numbers in parentheses indicate median paired-end distance for each sample (add 36 for both sequences and 50 for both Illumina adaptors (+172) to get complete library size).

Figure 3

Screen shots of ExpressionPlot 2way plot and table_browser. (a) 2way plot of human tissue panel RNA-Seq data [1] showing brain gene expression on the y-axis and average expression in all other tissues (pooled) on the x-axis. Blue points correspond to genes significantly higher (P ≤ 10^-4, fold change ≥20, 370 points) in brain relative to the other tissues; green points correspond to significantly lower. (b) 2way plot showing cassette exon usage (inclusion:skip read ratios) instead of gene levels in the same data set. The heavy lobe above the diagonal corresponds to exons with zero skipping reads in the brain, and the lighter lobe below the diagonal corresponds to exons with zero skipping reads in all other tissues. Although the P-values are still valid, in these regimes the inclusion:skip ratio statistic is less precise. (c) Partial screen shot of table browser showing brain-enriched cassette exons in the same data set. The context menu was triggered by the mouse clicking on the row for CLTA (clathrin, light chain A) and offers the user links to open the seqview genome browser tool in a window covering either the entire gene or just the alternative exon. In either case the exon will be automatically highlighted (Figure 5).

Figure 4

Screen shots of ExpressionPlot 4way plots showing cross-platform and cross-species comparisons. (a) Heart-enriched gene expression in human tissue panel exon array [28] (x-axis) and RNA-Seq [1] (y-axis) data sets. Points correspond to genes. Fold-change of expression in heart is plotted versus all other samples in corresponding data set. Genes enriched in heart are plotted further to the right (exon array) and/or up (RNA-Seq), and those higher in other samples are further to the left and/or down. Genes significantly different only on one platform are colored red (exon array) or green (RNA-Seq) and those different on both platforms are colored blue. P-value cutoffs are 0.01 for exon array and 10^-4 for RNA-Seq, and fold-change cutoffs are 2 for both platforms. Colored numbers show number of genes in each category. (b) Similar plot comparing the same x-axis (human heart-enriched gene expression by exon array) to mouse heart-enriched gene expression, also by exon array (y-axis).

Figure 5

Screen shot of ExpressionPlot's genome browser seqview. The region of the CLTA gene, which contains a brain-enriched exon (pink), is shown. Known transcripts of CLTA are seen along the bottom (arrowheads indicate plus strand). The accumulation of RNA-Seq reads from five human tissues is shown on the top. The heights of black bars indicate numbers of reads overlapping each genomic position, whereas the heights of blue brackets indicate numbers of reads overlapping splice junctions. Data from RNA-Seq human tissue panel [1].

Figure 6

ExpressionPlot screen shots examining spleen-enriched genes in human exon array tissue panel data [28]. (a) Levels of Myd88, a key signaling protein in the innate immune system [29], in human tissues using the genelev tool. (b) ecdf showing tissue enrichment (fold change relative to all other tissues) of the 316 genes least 5-fold enriched in the spleen at a P-value cutoff of 10^-4. The sharp angle at 2.3 in the spleen curve indicates the 5-fold cutoff. The position of the cerebellum curve to the left of all the others may reflect the general depletion of immune cells, which is characteristic of the spleen, within the nervous system. (c) event_heatmap showing the fold enrichments of the 316 spleen-enriched genes in all 11 tissues in the panel. The screen shot was edited by removing many of the genes from the middle for formatting purposes and adding an arrow to indicate Myd88, which is part of a cluster of spleen-enriched genes also enriched in the liver. The depletion of the spleen-enriched genes in the cerebellum is evident by the excess blue color in the cerebellum row.