Pre-sequencing assessment of RNA-Seq library quality using real-time qPCR

RNA sequencing (RNA-Seq) is an essential assay for studying transcriptome profiling. Ribosomal RNA (rRNA) comprises more than 80-90% of total cellular RNA; efficient removal is essential for accurately capturing transcriptomes, particularly to sequence low-abundance mRNAs. Inefficient rRNA removal during library preparation can result from variations in sample quality, preparation methods, and handling. Estimating rRNA content in RNA-Seq libraries pre-sequencing is therefore challenging due to the absence of a reliable and cost-effective assessment method. This study addresses the issue by introducing a scalable real-time quantitative polymerase chain reaction (qPCR) based assay targeting human 18S rRNA to evaluate rRNA depletion efficiency of RNA-Seq libraries pre-sequencing. qPCR efficiency was optimized using serial dilutions of Universal Human Reference (UHR) control, and Ct thresholds were established using pilot data from 644 libraries. Following this optimization, analysis of 1748 human Total RNA-Seq libraries and 445 Poly A + two widely used RNA-Seq library methods, demonstrated a strong correlation between 18S rRNA qPCR results and post-sequencing rRNA rates. This assay was also used to evaluate the performance of Oligo (dT) beads from four different vendors to enrich mRNA. This 18S rRNA qPCR assay is a cost-effective, scalable approach for reliably predicting rRNA read percentage in RNA-Seq libraries pre-sequencing.