CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5’ capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5’cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences. Long-read RNA sequencing has the potential to support exhaustive annotation of eukaryotic genomes, but reliable end-to-end sequencing is still challenging. Here the authors report a method called CapTrap-seq which combines the cap-trapping strategy with oligo(dT) priming for precise full-length transcript detection.