Gel-free library preparation for next-generation RNA sequencing and small RNA quantification

Abstract Next-generation RNA sequencing (RNA-seq) is hampered by “primer dimer” (PD) artifacts and its quantitative performance reduced by polymerase fall-off (PF) at RNA modifications and secondary structures. Here we improved RNA-seq efficiency by incorporating (i) a post-reverse-transcription (RT) digestion of excess primers with Escherichia coli exonuclease I for PD mitigation, thus obviating gel purification during RNA-seq library preparation, and (ii) a high-processivity reverse transcriptase to increase full-length reads. A full factorial experimental design was applied to absolute quantification RNA sequencing (AQRNA-seq), the most accurate NGS-based method for quantifying small RNAs, using cDNA libraries constructed from E. coli small RNAs (> 85% tRNA) followed by sequencing, data processing, and data analysis. The novel PF and PD mitigation approaches increased AQRNA-seq sensitivity > 10-fold by minimizing PF and maximizing target RNA reads. By increasing sensitivity and obviating gel electrophoresis for removing PD, AQRNA-seq and other NGS-based RNA-seq methods can now be automated to increase throughput and reduce RNA sample size.