Fusion gene identification by paired-end RNA-sequencing. (a) Identification of fusion gene candidates through selection of paired-end reads, the ends of which align to two different and non-adjacent genes. (b) Identification of the exact fusion junction by aligning non-mapped short reads against a computer generated database of all possible exon-exon junctions between the two partner genes. Separation of true fusions (left) from false positives (right) by examining the pattern of short read alignments across exon-exon junctions. Genuine fusion junctions are characterized by a stacked/ladder-like pattern of short reads across the fusion point. False positives lack this pattern; instead, all junction matching short reads align to the exact same position or are shifted by one to two base pairs. Furthermore, this alignment is mostly to one of the exons.