Abstract
A key component in many RNA-Seq based studies is contrasting multiple replicates from different experimental conditions. In this setup replicates play a key role as they allow to capture underlying biological variability inherent to the compared conditions, as well as experimental variability. However, what constitutes a “bad” replicate is not necessarily well defined. Consequently, researchers might discard valuable data or downstream analysis may be hampered by failed experiments. Here we develop a probability model to weigh a given RNA-Seq sample as a representative of an experimental condition when performing alternative splicing analysis. We demonstrate that this model detects outlier samples which are consistently and significantly different compared to other samples from the same condition. Moreover, we show that instead of discarding such samples the proposed weighting scheme can be used to downweight samples and specific splicing variations suspected as outliers, gaining statistical power. These weights can then be used for differential splicing (DS) analysis, where the resulting algorithm offers a generalization of the MAJIQ algorithm. Using both synthetic and real-life data we perform an extensive evaluation of the improved MAJIQ algorithm in different scenarios involving perturbed samples, mislabeled samples, no-signal groups, and different levels of coverage, showing it compares favorably to other tools. Overall, this work offers an outlier detection algorithm that can be combined with any splicing pipeline, a generalized and improved version of MAJIQ for differential splicing detection, and an evaluation pipeline researchers can use to evaluate which algorithm may work best for their needs.
