Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid-Phase Catalysis Approach.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Nanopore direct RNA sequencing requires substantial RNA input (500 ng), limiting low-input applications like pathogen surveillance.
• Current library preparation methods using solid-phase reversible immobilization (SPRI) beads cause significant sample loss, contributing to the high input requirement.

Purpose of the Study:

• To develop a novel RNA library preparation strategy for Nanopore direct RNA sequencing that minimizes sample input requirements.
• To overcome the limitations of SPRI bead purification in low-input RNA sequencing.

Main Methods:

• A solid-phase catalysis approach was developed, immobilizing poly(A) polymerase and T4 DNA ligase for concurrent processing of non-polyadenylated transcripts.
• The prepared library was directly loaded onto the Nanopore flow cell without SPRI bead purification.
• Whole transcriptome sequencing was performed using Listeria monocytogenes as a model organism.

Main Results:

• The novel method demonstrated minimal sample loss, reduced preparation time, and increased sequencing throughput compared to standard methods.
• Reduced nanopore fouling was observed, leading to higher quality sequencing data.
• The approach successfully enabled low-input Nanopore direct RNA sequencing, enhancing pathogen detection and transcript identification.

Conclusions:

• The solid-phase catalysis strategy significantly improves Nanopore direct RNA sequencing efficiency, particularly for low-input samples.
• This method facilitates improved pathogen surveillance and microbial community analysis.
• Accurate RNA consensus with high fidelity and identification of more expressed genes were achieved for both high and low RNA inputs.