Most Algorithms for Quantifying Full-length mRNA Splice Forms are Inaccurate

Because of the advantages of RNA sequencing (RNA-Seq) over microarrays, it is gaining widespread popularity for highly parallel gene expression analysis. For example, RNA-Seq is expected to be able to provide accurate identification and quantification of full-length splice forms. A number of informatics packages have been developed for this purpose, but short reads make it a difficult problem in principle. Sequencing error and polymorphisms add further complications. It has become necessary to perform studies to determine which algorithms perform best and which if any algorithms perform adequately. However, there is a dearth of independent and unbiased benchmarking studies. Here researchers at the University of Pennsylvania take an approach using both simulated and experimental benchmark data to evaluate their accuracy.


Accuracy results for simulated dataset T1 for the methods which utilize a reference genome. This represents the most ideal case where all genes are highly expressed, there are no polymorphisms and there are no alignment errors. Splicing is divided into three types, the only cases where precision was above 90% in the first two types are when there is a single splice form. The analysis was run with gene annotation provided.

Te researchers conclude that most methods are inaccurate even using idealized data, and that no method is highly accurate once multiple splice forms, polymorphisms, intron signal, sequencing errors, alignment errors, annotation errors, and other complicating factors are present. These results point to the pressing need for further algorithm development.

Availability: Simulated datasets and other supporting information can be found at


Hayer KE, Pizarro A, Lahens NF, Hogenesch JB, Grant GR. (2015) Benchmark Analysis of Algorithms for Determining and Quantifying Full-length mRNA Splice Forms from RNA-Seq Data. Bioinformatics [Epub ahead of print]. [article]

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