The qRT-PCR validation of differential splicing events detected by RNA-seq.

<p>qRT-PCR was performed for four genes that are identified as differential splicing genes between UCB and non-tumor tissues. The result of qRT-PCR is the relative expression level of the skipped exon and the neighboring constitutive exon. The expression level of each exon was normalized to the level in non-tumor tissue. The ΨMISO was the result of MISO, indicates the ratio of reads supporting inclusion exon vs. total reads supporting both inclusion and exclusion exon. A∼D: CD44, PDGFA, NUMB and GSK3B.</p

qRT-PCR validation of the differentially expressed genes in the additional patients.

<p>qRT-PCR was performed for five differentially expressed genes (CDH1, VEGFA, PTPRF, CLDN7 and MMP2) in the additional 11 patients (including 6 recurrent and drug-resistant UCB patients and 5 newly diagnosed patients). The histogram showed the proportion of validated patients in all cases (blue), the recurrent cases (red) and newly diagnosed (green).</p

RNA-Seq read mapping to the reference gene PDGFA.

<p>A: RNA-Seq read mapping to the UCSC reference genome (hg19) of the gene PDGFA for UCB and non-tumor tissues in this study. The UCB tracks are shown in red and non-tumor tissue in green. The pink band indicated the location of skipped exon. B: The detail of junction reads mapping to the skipped exon and its neighboring exons. The Ψ (”percentage spliced in”) indicates the ratio of reads supporting inclusion exon vs. total reads supporting both inclusion and exclusion exon. The Ψ posterior distributions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091466#pone.0091466-Katz1" target="_blank">[25]</a> were shown in the right side.</p

Statistics of alternative splicing events in bladder cancer.

<p>Statistics of alternative splicing events in bladder cancer.</p

Statistics of bladder cancer transcriptome mapping to human genome Hg19.

<p>#: Sequencing coverage on human transcriptome (approximately 113 million bps which was estimated as the total length of all unique exons according to Ensembl database).</p

The differentially expressed genes detected by RNA-seq are confirmed by qRT-PCR.

<p>qRT-PCR was performed for five genes that are identified as differential expressed genes between UCB and non-tumor tissues. The expression level of each gene was normalized to the level in non-tumor tissue. A-E: PTPRF, MMP2, VEGFA, CDH1 and CLDN7.</p

Statistics of differential splicing genes in bladder cancer.

<p>Statistics of differential splicing genes in bladder cancer.</p

Gene Ontology terms of enriched differentially expressed genes in bladder cancer.

<p>#: Fold Enrichment  =  (number of differentially expressed genes with the GO term/number of differentially expressed genes)/(number of expressed genes with the GO term/number of expressed genes)</p><p>*: p value corrected by method of Bonferroni, and only GO terms of the corrected p value less than 0.05 were shown.</p

KEGG pathways of enriched differentially expressed genes in bladder cancer.

#<p>: Fold Enrichment  =  (number of differentially expressed genes in the pathway/number of differentially expressed genes)/(number of expressed genes in the pathway/number of expressed genes)</p><p>*: False Discovery Rate provided by DAVID, only pathways of the FDR less than 0.05 were shown.</p

qRT-PCR validation of differential splicing events in the additional patients.

<p>qRT-PCR was performed for six differential splicing genes (CD44, PDGFA, NUMB, LPHN2, NIN and FAT1) in the additional 11 patients (including 6 recurrent and drug-resistant UCB patients and 5 newly diagnosed patients). The histogram showed the proportion of validated patients in all cases (blue), the recurrent cases (red) and newly diagnosed (green).</p