Long read sequencing reveals changes in measured isoform structure and abundance due to RNA processing

Direct RNA, direct cDNA, and PCR-amplified cDNA sequencing on seven SG-NEx cell line datasets show hundreds of differentially expressed genes and isoforms, with protocol-linked structural shifts.

• Why it matters: RNA preparation can bias measured expression and isoform structure, but RT-related effects are harder to quantify than PCR. This work addresses how protocol choice changes what nanopore sequencing reports.
• What they did: The study compared nanopore direct RNA, direct cDNA, and PCR-amplified cDNA across seven cell line datasets from SG-NEx, then analyzed differential expression, transcript usage, and isoform structural differences. It also examined m6A-linked changes and validated findings using raw coverage visualization.
• The result: Differentially expressed isoforms matched expected amplification biases, and PCR counts decreased with higher m6A methylation on native RNA. Most dominant isoform switches involved complex structural changes, and inserted introns (exitrons) were significantly more common after RT and amplification.

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