Figure 1.
Schematic representation of cDNA libraries.
An oligo-dT primer containing the DpnII restriction site was used for first-strand synthesis. Second-strand synthesis was performed with RNase H, DNA polymerase and T4 DNA ligase. The double-stranded cDNA was digested with DpnII, followed by coupling of linkers and PCR.
Figure 2.
Bioinformatics analyzes flowchart.
(A) Initial filters. (B) Human genome alignment. Completely Aligning to Human Genome corresponds to reads that aligned to the genome sequence using the criteria of coverage ≥70% and identity ≥96%. Sequences aligning to more than one genome region following these criteria were discarded. Single-hit high coverage genome alignment sequences were used for discovery of novel SNPs. Partially Aligning to Human Genome corresponds to reads that aligned to genome sequence using the criteria of coverage ≥20% and ≤80% and identity ≥99.9%. These reads were used for discovery of gene fusion events. (C) Transcript databases alignment. For transcriptome analysis, reads from completely aligning sub-set were further aligned to known gene databases for the discovery of novel splicing variants. Additionally, reads were aligned to RefSeq databases for obtaining the number of transcripts (mRNAs) and genes identified.
Figure 3.
Genomic coordinates of the partial sequences of novel human genes.
Arrows represent the genomic localization of each gene and its transcription orientation. The red arrows represent the novel genes [(A) NG7; (B) NG8; (C) NG9]. In an expanded view, the genomic coordinates of the NGs are shown, as well as the conserved splice sites depicted in the introns and the DpnII restriction sites. Genomic representations are not scaled.
Figure 4.
Putative new alternative splicing variants -
The 2,865 novel alternative splicing events detected in our approach are distributed according to the type of event reported. White squares represent the known exons and grey squares represent the alternative exons. The number of events is shown on the right side of each event type. (A) Intron retention showing the presence of one or more constitutive exons. (B) Alternative splice donor or acceptor site usage. (C) Alternative exon usage events were sub-classified into exon skipping and exon inclusion events that show one or both flanking constitutive exons.
Figure 5.
Identification and validation of gene fusion events.
(A) Circus-plot representation of inter- and intra-chromossomal gene fusion identified for each cell line, C5.2 (left panel) and HB4a (right panel), (*) Inter-chromosomal gene fusion events reported exclusively by one of the cell lines. (B) Validation of 3 gene fusions. The exon distribution of the original genes is represented by the numbered squares, and the regions involved in the fusion are represented by the colored lines.
Figure 6.
(A) Correlation of Unigene cluster sizes classified by the ESTs abundance-classes: bottom 25%, 10% and 5% (left panel) and top 25%, 10% and 5% (right panel) by the average number of reads from the RNAseq (log10). (B) Correlation between number of reads of RNA-seq and Cycle threshold (Ct values) of qRT-PCR experiments for all, validated and non-validated gene set. (C) Relative gene expression between C5.2 and HB4a cells. The 2 black lines represent the cut-off value of log2 ratio ≥|2|- fold-change ≥|4|. The blue colored points correspond to genes with a Bayes Error Rate equal to 0.0. ERBB2 relative expression is identified by the red point. (D) Classification of breast tumor samples in high and basal ERBB2-expression by relative quantification of ERBB2 transcript. The black circled points represent the expression level of the cell lines with high (C5.2) and basal (HB4a) ERBB2 expression levels. (E) Relative expression of the genes LOX, MME, GALNT3 and ATP5L in the two groups of tumor breast samples with high and basal ERBB2 expression.