Systematic assessment of RNA-Seq experimental workflows for plasma cell-free transcriptome profiling

The use of plasma cell-free RNA (cfRNA) as a diagnostic biomarker in liquid biopsies, while promising, presents important technical challenges. These include the acquisition of high-quality, diverse cfRNA molecules, mitigating genomic DNA contamination in sequencing libraries, and the optimization of bioinformatics pipelines. In addition, there is currently no gold-standard workflow for the profiling of plasma cfRNA. A systematic large-scale comparison of cfRNA-Seq experimental workflows is thus needed to address these issues. In this study, we present a first-of-its-kind comprehensive, systematic meta-analysis of publicly available plasma cfRNA-Seq datasets, which we re-analyzed using a uniform bioinformatics pipeline. We compare various analytical metrics across over 2,500 samples from a comprehensive, curated list of 19 published peer-reviewed cfRNA-Seq studies. Our results show how details of the experimental protocol influence cfRNA-Seq data quality in often drastic ways, leading to considerable variability and batch effects across studies. Such a benchmark contributes to the advancement of cfRNA-based diagnostic methods and sets the stage for future improvements and standardizations of cfRNA profiling workflows.