Deciphering transcriptome complexity via long‐read sequencing

Abstract

Abstract Transcriptomics is moving beyond gene‐level quantification toward isoform‐resolved interrogation of alternative splicing, transcript structural variation, and repeat‐derived transcription. Yet short‐read sequencing remains intrinsically limited in accurately reconstructing full‐length transcripts and resolving complex repetitive regions, including transposable elements. Recent advances in long‐read sequencing, exemplified by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), offer a transformative opportunity to directly observe complete RNA molecules and thereby overcome these bottlenecks. However, practical adoption is hindered by demanding library construction and the need to process noisy, fast‐evolving long‐read data, and the field still lacks a unified resource that guides researchers through the entire experimental and analytical workflow. This review fills that gap by providing a concise, end‐to‐end, and implementation‐oriented roadmap for long‐read transcriptomics. We distill the key decisions from platform and library strategy selection to core computational processing and downstream interpretation, and we summarize emerging frontiers and best‐practice recommendations. By offering a reusable framework and practical checklists, this guide empowers a broader community to exploit long reads for standardized, reproducible, isoform‐level discovery at unprecedented resolution.