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Alternatively spliced transcript isoforms are commonly observed in higher eukaryotes. The expression levels of these isoforms are key for understanding normal functions in healthy tissues and the progression of disease states. However, accurate quantification of expression at the transcript level is limited with current RNA-seq technologies because of, for example, limited read length and the cost of deep sequencing.

A large number of tools have been developed to tackle this problem, and we performed a comprehensive evaluation of these tools using both experimental and simulated RNA-seq datasets. We found that recently developed alignment-free tools are both fast and accurate. The accuracy of all methods was mainly influenced by the complexity of gene structures and caution must be taken when interpreting quantification …