Analyzing RNA-Seq Data fromChlamydiawith Super Broad Transcriptomic Activation: Challenges, Solutions, and Implications for Other Systems

Abstract

AbstractMotivationRNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes ofChlamydiaspp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends.ResultsBy incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. While the strategies employed are developed in the context ofChlamydia, the principles of flexible and context-aware normalization may inform adjustments in other systems with unbalanced gene expression dynamics, such as bacterial spore germination.Availability and implementationThe code for reproducing the presented bioinformatic analysis is available athttps://zenodo.org/records/11201379.