Abstract
The requirement of a large input amount (500 ng) for Nanopore direct RNA-seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid-phase reversible immobilization (SPRI) beads. A novel solid-phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non-polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low-input direct RNA-seq, improving pathogen detection and transcript identification in a microbial community standard with spike-in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.