A systematic benchmark of nanopore long-read RNA sequencing for transcript-level analysis in human cell lines

Chen, Davidson, Wan, and Yao et al. present their results from the Singapore Nanopore Expression (SG-NEx) project, in which they compared five RNA sequencing protocols across a benchmark dataset from seven human cell lines. The authors report that Oxford Nanopore sequencing offers superior detection of major isoforms, novel transcripts, and RNA modifications compared to other sequencing methods, providing a valuable resource for advancing transcriptomic research.

Key points:

• Short-read RNA sequencing introduces fragmentation and biases, making it difficult to accurately identify full-length RNA transcripts — critical for understanding gene regulation and disease mechanisms

• The authors compared Oxford Nanopore direct RNA, direct cDNA and PCR-amplified cDNA kits, Illumina cDNA, and PacBio IsoSeq

• Oxford Nanopore sequencing better captured full-length transcripts and alternative isoforms compared to Illumina short-read sequencing

• The direct RNA and cDNA kits performed best at detecting novel transcripts, fusion genes, and RNA modifications

• Oxford Nanopore direct RNA sequencing was unique in its ability to sequence native RNA, avoiding biases from cDNA conversion and PCR amplification

Sample type: human cell lines

Kit: Direct RNA, Direct cDNA, PCR cDNA