Distinct 5' and 3' Coverage Biases Shape Transcriptome Interpretation in Nanopore Direct RNA versus PCR-cDNA Sequencing

Long-read RNA sequencing enables isoform-resolved transcriptomics, but library preparation introduces systematic biases that shape biological interpretation. We benchmarked Oxford Nanopore's two protocols—PCR-cDNA and direct RNA—using SKMM2 myeloma cells stimulated with interleukin-6 (IL-6) and ERCC synthetic spike-ins. Direct RNA produced longer, higher-quality reads and more high-confidence isoforms, but showed pronounced 5' coverage loss. PCR-cDNA yielded shorter fragments with 3' underrepresentation, detecting more low-abundance transcripts at reduced confidence. Protocol-specific biases had major consequences: differential expression analysis revealed limited overlap in IL-6–responsive genes, and pathway enrichment was broader in direct RNA. At the isoform level, differential transcript usage was almost entirely protocol-specific, with case studies (e.g. RPL22L1, GRB2, RNF220) illustrating concordance and divergence. ERCC controls confirmed these biases as technical rather than biological. Together, our results show that while both methods provide accurate gene-level quantification, transcript-level conclusions depend critically on protocol choice, highlighting the need for careful selection in long-read transcriptomics. Overall design: SKMM2 multiple myeloma cells were cultured and treated in biological triplicates with either interleukin-6 (IL-6, 1 ng/mL, 18 h) or vehicle control (0.2% BSA). Total RNA was extracted and used to prepare sequencing libraries with two Oxford Nanopore Technologies (ONT) protocols: (i) PCR-cDNA sequencing (SQK-PCB111.24, 14 PCR cycles for SKMM2 RNA, 16 cycles for ERCC spike-ins) and (ii) direct RNA sequencing (SQK-RNA004). Each protocol was applied to both IL-6–treated and control samples, enabling direct benchmarking of protocol-specific biases. ERCC synthetic RNA spike-ins were sequenced in parallel as external controls. Libraries were sequenced on ONT PromethION flow cells (R9.4.1 for PCR-cDNA, RNA flow cells for direct RNA) with live base-calling. Downstream analyses included gene- and isoform-level quantification, differential expression, pathway enrichment, and differential transcript usage.