Abstract: Methods, systems, and software are provided for detecting gene fusions in a subject with a cancer condition through mRNA boundary analysis of next generation sequencing of a transcriptome or relevant part thereof. Methods, systems, and software are provided for detecting splice variants in a subject with a cancer condition through mRNA boundary analysis of next generation sequencing of a transcriptome or relevant part thereof. Methods, systems, and software are provided for evaluating the complexity of an RNA-seq sequencing reaction through mRNA boundary analysis. Generally, the methods described herein include obtaining sequences of mRNA molecules for a plurality of genes in a sample of a subject. For each gene, an RNA boundary distribution including relative abundance value for each respective RNA boundary sub-sequence of the gene is determined from the plurality of sequences. These abundance values are evaluated using one or more models to provide the analyses described herein.