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Research: Nanopore direct RNA sequencing reveals METTL2A-mediated m³C sites in poly(A) RNA
- Shuhei Mitsutomi,
- Anzu Sugawara,
- Masahide Seki,
- Yutaka Suzuki,
- Sotaro Miyao,
- Haozhe Du,
- Kenji Takahashi,
- Yusuke Mizukami,
- Kenzui Taniue,
- and Nobuyoshi Akimitsu
Genome Res. November 2025 35: 2406-2417; Published in Advance October 30, 2025, doi:10.1101/gr.280269.124
...motif and coding sequences. METTL2A knockdown alters expression of S100A4 mRNA isoforms, which contains METTL2A-mediated m3C sites. Notably, many transcripts with METTL2A-mediated m3C sites are upregulated upon METTL2A knockdown. We reveal the transcriptome-wide presence of m3C sites in poly(A) RNA...
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Method: Deep structural clustering reveals hidden systematic biases in RNA sequencing data
- Qiang Su,
- Yi Long,
- Deming Gou,
- Junmin Quan,
- Xiaoming Zhou,
- and Qizhou Lian
Genome Res. November 2025 35: 2563-2577; Published in Advance September 19, 2025, doi:10.1101/gr.280713.125
...using RNA-seq data from six colorectal samples. A stacking plot (Fig. 6A) comparing the 50-mer sequencing count profiles of the EMP1-211 transcript revealed significant variability in coverage patterns. These findings suggest that full-length transcript coverage is shaped by both global isoform...
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Method: Analysis of coding gene expression from small RNA sequencing
- Aygun Azadova,
- Anthonia Ekperuoh,
- Greg N. Brooke,
- and Antonio Marco
Genome Res. March 2026 36: 611-618; Published in Advance February 10, 2026, doi:10.1101/gr.281364.125
...–target pairing is partially complementary, as is typically the case in animals, protein synthesis is repressed (Bartel 2009). However, target RNA stability is also influenced by miRNA targeting, and particularly for miRNAs exerting strong regulatory effects, target degradation is expected (Baek et al. 2008...
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Method: Long-read RNA sequencing reveals allele-specific N⁶-methyladenosine modifications
- Dayea Park and
- Can Cenik
Genome Res. April 2025 35: 999-1011; Published in Advance October 29, 2024, doi:10.1101/gr.279270.124
...-methyladenosine (m6A) modifications in native mRNA. We used human and mouse cells with known genetic variants to assign the allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses reveal the importance of sequences adjacent...
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Method: Long-read subcellular fractionation and sequencing reveals the translational fate of full-length mRNA isoforms during neuronal differentiation
- Alexander J. Ritter,
- Jolene M. Draper,
- Christopher Vollmers,
- and Jeremy R. Sanford
Genome Res. November 2024 34: 2000-2011; Published in Advance June 5, 2024, doi:10.1101/gr.279170.124
...localization, stability, and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESCs) and neural progenitor...
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Resource: Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms
- José Carlos Montañés,
- Marta Huertas,
- Simone G. Moro,
- William R. Blevins,
- Mercè Carmona,
- José Ayté,
- Elena Hidalgo,
- and M. Mar Albà
Genome Res. June 2022 32: 1215-1227; Published in Advance May 26, 2022, doi:10.1101/gr.276516.121
...were expressed at much lower levels than mRNAs (average 70 dRNA reads vs. 1130 dRNA reads).We inspected the correlation between dRNA and Illumina derived transcript abundances. Whereas each dRNA read corresponds to one native molecule, Illumina sequencing involves cDNA synthesis and PCR amplification...
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Research: Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing
- Karine Choquet,
- Louis-Philippe Chaumont,
- Simon Bache,
- Autum R. Baxter-Koenigs,
- and L. Stirling Churchman
Genome Res. April 2025 35: 712-724; Published in Advance February 14, 2025, doi:10.1101/gr.279203.124
...the many introns of a human gene are removed can substantially influence AS, while nascent RNA polyadenylation can affect RNA stability and decay. However, how splicing order and poly(A) tail length are regulated by genetic variation has never been explored. Here, we used direct RNA nanopore sequencing...
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Research: Deciphering the largest disease-associated transcript isoforms in the human neural retina with advanced long-read sequencing approaches
- Merel Stemerdink,
- Tabea Riepe,
- Nick Zomer,
- Renee Salz,
- Michael Kwint,
- Jaap Oostrik,
- Raoul Timmermans,
- Barbara Ferrari,
- Stefano Ferrari,
- Alfredo Dueñas Rey,
- Emma Delanote,
- Suzanne E. de Bruijn,
- Hannie Kremer,
- Susanne Roosing,
- Frauke Coppieters,
- Alexander Hoischen,
- Frans P.M. Cremers,
- Peter A.C. ‘t Hoen,
- Erwin van Wijk,
- and Erik de Vrieze
Genome Res. April 2025 35: 725-739; Published in Advance March 4, 2025, doi:10.1101/gr.280060.124
...(IRDs) like Usher syndrome, which involves 11 associated genes with transcripts up to 19.6 kb. To address this, we used PacBio long-read mRNA isoform sequencing (Iso-Seq) following standard library preparation and an optimized workflow to enrich for long transcripts in the human neural retina. While our...
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Method: The SeqSplice multiplexed minigene splicing assay for characterization and quantitation of variant-induced BRCA1 and BRCA2 splice isoforms
- Daffodil M. Canson,
- Michael T. Parsons,
- Gemma Moir-Meyer,
- Troy Dumenil,
- Gemma Montalban,
- Erica Lin,
- Terri P. McVeigh,
- Aimee L. Davidson,
- Shaun M. Bouckaert,
- Matt Trau,
- Darren Korbie,
- and Amanda B. Spurdle
Genome Res. September 2025 35: 2104-2115; Published in Advance August 6, 2025, doi:10.1101/gr.279557.124
..., and BRCA1 Δ10 and BRCA2 Δ2, Δ4, and Δ6 are minor alternative isoforms detected in clinically relevant samples (Colombo et al. 2014; Fackenthal et al. 2016). BRCA2 Δ7 is also a natural isoform detected in 1% of RNA sequencing samples in SpliceVault (Dawes et al. 2023). Naturally occurring BRCA1 alternative...
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Method: Detecting m6A RNA modification from nanopore sequencing using a semisupervised learning framework
- Haotian Teng,
- Marcus Stoiber,
- Ziv Bar-Joseph,
- and Carl Kingsford
Genome Res. November 2024 34: 1987-1999; Published in Advance October 15, 2024, doi:10.1101/gr.278960.124
.... 2014; Murakami and Jaffrey 2022).High-throughput sequencing from Illumina, also known as sequencing by synthesis (SBS), identifies nucleotides through synthesis, leading to the loss of posttranscriptional information (Buermans and Den Dunnen 2014). Therefore, indirect methods are required to detect RNA...
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