Abstract
We report the first quantitative and qualitative analysis of the poly (A)
+
transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in
ERBB2
over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as
LOX
,
ATP5L
,
GALNT3
, and
MME
revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different
ERBB2
backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by
ERBB2
overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.