2025/08/24 by Felix Pförtner, Eva Briem, Wolfgang Enard +1 · 3 voices · 1 citation
Biochemistry, Genetics and Molecular Biology · #Cancer-related molecular mechanisms research #Molecular Biology Techniques and Applications
paper · pdf · doi:10.1101/2025.08.20.671269
openalex publication_date 2025/08/24 · openalex created_date 2025/08/25 · openalex updated_date 2026/07/15
Abstract Bulk and single-cell RNA-seq are powerful tools for transcriptomic analysis, providing insights into many aspects of molecular and cellular phenotypes. Costs constrain the amount of biological insight obtainable within a given budget, and as sequencing prices decline, efficient library protocols have become a decisive factor. In this study, we introduce an approach to systematically optimize the number of usable reads that RNA-seq protocols generate. We applied this ”funnel strategy” to prime-seq, an early-barcoding bulk RNA-seq protocol, by systematically testing critical protocol steps totaling 1080 samples in 49 libraries. This resulted in the optimized prime-seq2 protocol that increases the number of usable reads by 60 % and improves one of the most cost-efficient bulk RNA-seq protocols available. Our study also suggests that monitoring the filtering of usable reads can serve as a valuable quality control for many RNA-seq protocols and sheds light on the complexity of the conditions and interactions that shape RNA-seq library composition and their interpretation.