Local sequence and sequencing depth dependent accuracy of RNA-seq reads

Background: Many biases and spurious effects are inherent in RNA-seq technology, resulting in a non-uniform distribution of sequencing read counts for each base position in a gene. Therefore, a base-level strategy is required to model the non-uniformity. Also, the properties of sequencing read counts can be leveraged to achieve a more precise estimation of the mean and variance of measurement. Results: In this study, we aimed to unveil the effects on RNA-seq accuracy from multiple factors and develop accurate modeling of RNA-seq reads in comparison. We found that the overdispersion rate decreased when sequencing depth increased on the base level. Moreover, the influence of local sequence(s) on the overdispersion rate was notable but no longer significant after adjusting the effect from sequencing depth. Based on these findings, we propose a desirable beta-binomial model with a dynamic overdispersion rate on the base-level proportion of sequencing read counts from two samples. Conclusions: The current study provides thorough insights into the impact of overdispersion at the position level and especially into its relationship with sequencing depth, local sequence, and preparation protocol. These properties of RNA-seq will aid in improvement of the quality control procedure and development of statistical methods for RNA-seq downstream analyses.