Accurate detection of differential RNA processing

rDiffDeep transcriptome sequencing (RNA-Seq) has become a vital tool for studying the state of cells in the context of varying environments, genotypes and other factors. RNA-Seq profiling data enable identification of novel isoforms, quantification of known isoforms and detection of changes in transcriptional or RNA-processing activity. Existing approaches to detect differential isoform abundance between samples either require a complete isoform annotation or fall short in providing statistically robust and calibrated significance estimates.

Now, a team led by researchers from Computational Biology Center, Sloan-Kettering Institute and the Friedrich Miescher Laboratory of the Max-Planck Society have developed a suite of statistical tests to address these open needs that includes:

  • A statistical test called rDiff.parametric that based on the gene annotation, detects differential splicing. This test models the counts in a similar way as DESeq and can account for biological variance.
  • A statistical test called rDiff.nonparametric that tests for a difference in the read distribution at a genomic locus. Therefore, it only needs the gene start and stop to detect detects differential relative transcript abundance and can be used when the splicing events are not yet annotated. This test is based on the Maximum mean discrepancy test and can also account for biological variance.

The team demonstrates:

1) the statistical calibration of the proposed is better than the one of CuffDiff (although not perfect) .

2) rDiff.parametric outperforms MISO and CuffDiff on a realistic simulated dataset.

3) rDiff.nonparametric has a similar performance as the Miso and CuffDiff without using the gene annotation for testing.

4) the methods perform well on two real datasets (from A.thaliana and D.melanogaster).

Availability – The proposed toolkit is available from and enables in-depth analyses of transcriptomes, with or without available isoform annotation.

  • Drewe P, Stegle O, Hartmann L, Kahles A, Bohnert R, Wachter A, Borgwardt K, Rätsch G. (2013) Accurate detection of differential RNA processing. Nucleic Acids Res [Epub ahead of print]. [article]
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