Increasing usable reads in RNA-seq protocols

Bulk and single-cell RNA-seq are powerful tools for transcriptomic analysis, providing insights into molecular and cellular phenotypes. Costs constrain the biological insights 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 generated by RNA-seq protocols. We applied this "funnel strategy" to prime-seq, an early-barcoding bulk RNA-seq protocol, by systematically testing critical protocol steps totaling 1,256 samples in 65 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 usable reads can serve as a valuable quality control for many RNA-seq protocols and sheds light on the complex interplay of experimental conditions that shape RNA-seq library composition and their interpretation.