DEMINERS enables clinical metagenomics and comparative transcriptomic analysis by increasing throughput and accuracy of nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) is a powerful tool for RNA biology but suffers from low basecalling accuracy, low throughput, and high input requirements. We present DEMINERS, a novel DRS toolkit combining an RNA multiplexing workflow, a Random Forest-based barcode classifier, and an optimized convolutional neural network basecaller with species-specific training. DEMINERS enables accurate demultiplexing of up to 24 samples, reducing RNA input and runtime. Applications include clinical metagenomics, cancer transcriptomics, and parallel transcriptomic comparisons, uncovering microbial diversity in COVID-19 and m(6)A's role in malaria and glioma. DEMINERS offers a robust, high-throughput solution for precise transcript and RNA modification analysis.