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Method: 3′-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors
- Jacob Stewart-Ornstein,
- Matthew J. Irby,
- Marina K. Lilieholm,
- Dylan Laprise,
- Maria D. Collier,
- Thomas Aunins,
- Dewi Harjanto,
- Aaron N. Chang,
- Deepak Reyon,
- and Jeremy S. Duffield
Genome Res. September 2025 35: 2064-2075; Published in Advance August 5, 2025, doi:10.1101/gr.280164.124
...Following the ligation reaction, samples are enzymatically fragmented to the appropriate size for Illumina sequencing (∼200–800 bp) by adding 2 µL of NEBNext dsDNA fragmentase in 1× fragmentation buffer (NEB) to final volume of 50 µL. The fragmentation reaction is incubated for 1 h at 37°C, terminated by adding 10 µ...
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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
...I restriction sites of pcDNA3.1 C-FLAG using the Ligation-Convenience kit (Nippon Gene, #315-05963). The base editor construct targeting the S100A4-201 m3C sites was generated by cloning a guide RNA (gRNA) sequence—designed to target the 5′ region of the third exon of S100A4-201—into the BbsI restriction sites...
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Method: Verkko2 integrates proximity-ligation data with long-read De Bruijn graphs for efficient telomere-to-telomere genome assembly, phasing, and scaffolding
- Dmitry Antipov,
- Mikko Rautiainen,
- Sergey Nurk,
- Brian P. Walenz,
- Steven J. Solar,
- Adam M. Phillippy,
- and Sergey Koren
Genome Res. July 2025 35: 1583-1594; Published in Advance May 19, 2025, doi:10.1101/gr.280383.124
...for segmental duplications. Translocations (Ohno et al. 1961) and ongoing recombination (Guarracino et al. 2023) within the short arms have maintained a high sequence similarity across all five acrocentric chromosomes (Nurk et al. 2022). The rDNA arrays can stretch for megabases, exceeding the length of current...
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Research: Atypical epigenetic and small RNA control of degenerated transposons and their fragments in clonally reproducing Spirodela polyrhiza
- Rodolphe Dombey,
- Daniel Buendía-Ávila,
- Verónica Barragán-Borrero,
- Laura Diezma-Navas,
- Arturo Ponce-Mañe,
- José Mario Vargas-Guerrero,
- Rana Elias,
- and Arturo Marí-Ordóñez
Genome Res. March 2025 35: 522-544; Published in Advance March 4, 2025, doi:10.1101/gr.279532.124
...modifying complexes to TEs through sequence complementarity (Malone and Hannon 2009; Moazed 2009). In flowering plants (angiosperms), TE silencing is mostly associated with DNA methylation and H3K9me2, and the mechanism by which sRNAs guide chromatin modifications is known as RNA-directed DNA methylation...
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Method: An optimized protocol for quality control of gene therapy vectors using nanopore direct RNA sequencing
- Kathleen Zeglinski,
- Christian Montellese,
- Matthew E. Ritchie,
- Monther Alhamdoosh,
- Cédric Vonarburg,
- Rory Bowden,
- Monika Jordi,
- Quentin Gouil,
- Florian Aeschimann,
- and Arthur Hsu
Genome Res. November 2024 34: 1966-1975; Published in Advance October 28, 2024, doi:10.1101/gr.279405.124
...(ONT) direct cDNA (Sertkaya et al. 2021) approaches have both been used to demonstrate improvements to lentiviral vectors (LVs), and sequencing has also been applied to other nucleic acid-based therapeutics, such as mRNA vaccines (Gunter et al. 2023). However, there is currently no clear consensus...
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Research: Strong bias in long-read sequencing prevents assembly of Drosophila melanogaster Y-linked genes
- A. Bernardo Carvalho,
- Bernard Y. Kim,
- and Fabiana Uno
Genome Res. January 2026 36: 71-82; Published in Advance October 1, 2025, doi:10.1101/gr.280604.125
...the proportion of each region's sequence occupied by non-B DNA motifs. Blue bars represent low-coverage Y-linked regions; black bars correspond to normal-coverage Y-linked regions and the euchromatic control region (white gene). Note that mirror repeats and direct repeats are consistently abundant in low...
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Method: Rapid and accurate demultiplexing of direct RNA nanopore sequencing data with SeqTagger
- Leszek P. Pryszcz,
- Gregor Diensthuber,
- Laia Llovera,
- Rebeca Medina,
- Anna Delgado-Tejedor,
- Luca Cozzuto,
- Julia Ponomarenko,
- and Eva Maria Novoa
Genome Res. April 2025 35: 956-966; Published in Advance January 29, 2025, doi:10.1101/gr.279290.124
...leads to robust and accurate demultiplexing statistics, it is computationally expensive, limited to four barcodes, and does not support newer RNA004 kit chemistries.We hypothesized that training a DNA basecaller that could directly basecall a DNA barcode sequence present in the RTA, which is ligated...
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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
...peptides to the immune system (Voorter et al. 2016), it is critical to understand how polymorphisms impact HLA pre-mRNA maturation. However, due to the technical challenges associated with analyzing HLA transcripts using short-read RNA-seq reads, HLA genetic variation has been primarily studied at the DNA...
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Method: Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling
- Li-Ting Chen,
- Myrthe Jager,
- Dàmi Rebergen,
- Geertruid J. Brink,
- Tom van den Ende,
- Willem Vanderlinden,
- Pauline Kolbeck,
- Marc Pagès-Gallego,
- Ymke van der Pol,
- Nicolle Besselink,
- Norbert Moldovan,
- Nizar Hami,
- Wigard P. Kloosterman,
- Hanneke van Laarhoven,
- Florent Mouliere,
- Ronald Zweemer,
- Jan Lipfert,
- Sarah Derks,
- Alessio Marcozzi,
- and Jeroen de Ridder
Genome Res. April 2025 35: 886-899; Published in Advance January 13, 2025, doi:10.1101/gr.279144.124
...are reduced by the consensus of multiple (≥3) repeats. NanoRCS offers a precise, multimodal nanopore sequencing-based strategy for cfDNA sequencing through the accurate identification of tumor-informed SNVs along with CNAs and fragmentation length patterns in cfDNA (Fig. 1D).View larger version...
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Research: Long-read sequencing reveals HBV integration patterns and oncogenic impact on early-onset hepatocellular carcinoma
- Yao Wang,
- Dong Yu,
- Yue Mei,
- ZhiDa Fu,
- Jian Lin,
- Di Wu,
- Yuan Yang,
- and Hongli Yan
Genome Res. November 2025 35: 2389-2405; Published in Advance September 16, 2025, doi:10.1101/gr.279889.124
...randomly to 8 kb using a g-tube. Nick repair (NEB next FFPE DNA repair mix), dA tailing (NEB next Ultra II end repair/dA-tailing module), and adapter ligation (NEB next blunt/TA ligase master mix) were sequentially performed on the sheared gDNA fragments, and DNA purification was performed between each...

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