An rRNA-depleted full-length transcriptome strategy using nanopore sequencing for identification of novel lncRNA isoforms

Long non-coding RNAs (lncRNAs) play crucial roles in gene regulation, but their full-length isoforms are often missed because of the limitations of poly(A)-based enrichment and short-read sequencing. Here, we aim to establish a comprehensive transcriptome profiling that captures both poly(A)+ and poly(A)- RNA isoforms using Oxford Nanopore Technologies (ONT) R10.4.1 flowcells. We establish an rRNA-depleted full-length transcriptome sequencing workflow (NanoncRNA-Seq), and use it together with Illumina NovaSeq to profile lncRNA isoforms in Saccharomyces cerevisiae under glucose and ethanol-associated physiological states. We combine multiple analytical tools to evaluate expression levels, splicing patterns, variants, and lncRNA identification. ONT sequencing achieves high accuracy (Q-score: 22.35, 99.42%) and detects fewer SNP and more novel isoforms, while Illumina sequencing reports fewer INDELs. Expression profiles are highly consistent within each platform and moderately across platforms. Notably, NanoncRNA-seq enables isoform-resolved lncRNA discovery and recoveres substantially more lncRNAs than Illumina (Pinfish: n = 260; Illumina: n = 51), including more lincRNAs (n = 201 vs. n = 25), likely because low-abundance transcripts are difficult to reconstruct from short reads. Overall, NanoncRNA-Seq effectively captures full-length lncRNA isoform discovery and highlights the complementary strengths of ONT in transcriptome research. This work describes NanoncRNA-Seq, an rRNA-depleted ONT workflow capturing full length poly(A)+/poly(A)- lncRNA isoforms in yeast.