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Method: High-resolution spatial transcriptomics in fixed tissue using a cost-effective PCL-seq workflow
- Xue Dong,
- Mengzhu Hu,
- Xiaonan Cui,
- Wenjian Zhou,
- Jingtao Cai,
- Guangyao Mao,
- and Weiyang Shi
Genome Res. September 2025 35: 2052-2063; Published in Advance August 5, 2025, doi:10.1101/gr.279906.124
.... In nonilluminated areas, the PC linker blocked adapter ligation, ensuring that only cDNA from illuminated regions was amplified and identified with high-throughput sequencing (Fig. 1).View larger version: In this window In a new window Figure 1. Schematic of the light-controlled DNA attachment strategy. Frozen...
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Method: Tissular chromatin-state cartography based on double-barcoded DNA arrays that capture unloaded PA-Tn5 transposase
- Maria Grazia Mendoza-Ferri,
- Gwendoline Lozachmeur,
- Maximilien Duvina,
- Laetitia Perret,
- Didier Merciris,
- Anne Gigout,
- and Marco Antonio Mendoza-Parra
Genome Res. July 2025 35: 1633-1645; Published in Advance May 13, 2025, doi:10.1101/gr.280305.124
...by magnesium chloride, leading to the formation of hairpin-like DNA structures. (E) Scheme illustrating the molecular biology steps required for generating a copy of the genomic DNA captured by the DNA probes, followed by their amplification for Illumina massive parallel DNA sequencing.Printed DNA arrays...
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Method: CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes
- Marzia Rossato,
- Luca Marcolungo,
- Luca De Antoni,
- Giulia Lopatriello,
- Elisa Bellucci,
- Gaia Cortinovis,
- Giulia Frascarelli,
- Laura Nanni,
- Elena Bitocchi,
- Valerio Di Vittori,
- Leonardo Vincenzi,
- Filippo Lucchini,
- Kirstin E. Bett,
- Larissa Ramsay,
- David James Konkin,
- Massimo Delledonne,
- and Roberto Papa
Genome Res. May 2023 33: 787-797; Published in Advance May 1, 2023, doi:10.1101/gr.277628.122
.... This could be achieved by using enzymes that enable target enrichment by depleting unwanted sequences from HTS libraries. For example, the duplex-specific nuclease (DSN) selectively digests double-stranded DNA molecules, and can be used to eliminate highly abundant sequences in a controlled denaturation...
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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
...with OVCA (Fig. 1E; Supplemental Table S1). NanoRCS libraries of cfDNA were sequenced on nanopore MinION and/or PromethION systems. To allow comparison with Illumina sequencing, shallow whole- sequencing (WGS) with NovaSeq was performed in parallel on all tumor cfDNA samples and three of the HC samples...
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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
.... As there was no detectable difference between the proportions of transcripts produced by the different input amounts of RNA/cDNA, this enabled more effective high-throughput screening, as the workflow did not require normalization of cDNA amounts prior to PCR. Our control experiment also addressed the potential differences...
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Method: High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics
- Maurizio Pellegrino,
- Adam Sciambi,
- Sebastian Treusch,
- Robert Durruthy-Durruthy,
- Kaustubh Gokhale,
- Jose Jacob,
- Tina X. Chen,
- Jennifer A. Geis,
- William Oldham,
- Jairo Matthews,
- Hagop Kantarjian,
- P. Andrew Futreal,
- Keyur Patel,
- Keith W. Jones,
- Koichi Takahashi,
- and Dennis J. Eastburn
Genome Res. September 2018 28: 1345-1352; Published in Advance August 7, 2018, doi:10.1101/gr.232272.117
...and enabling efficient amplification in the presence of high concentrations of crude lysate (Jackson et al. 1990; Strauss 2001).In this report, we present a microfluidic approach, relying on cell-identifying molecular barcodes, that overcomes existing barriers to high-throughput single-cell DNA sequencing. We...
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Method: A generic, cost-effective, and scalable cell lineage analysis platform
- Tamir Biezuner,
- Adam Spiro,
- Ofir Raz,
- Shiran Amir,
- Lilach Milo,
- Rivka Adar,
- Noa Chapal-Ilani,
- Veronika Berman,
- Yael Fried,
- Elena Ainbinder,
- Galit Cohen,
- Haim M. Barr,
- Ruth Halaban,
- and Ehud Shapiro
Genome Res. November 2016 26: 1588-1599; Published in Advance August 24, 2016, doi:10.1101/gr.202903.115
...on the flanking universal sequence to attach a sample-specific barcode and to form a full-length Illumina library. In order to validate the two-step PCR scheme, we processed SC DNA samples that originated from mismatch repair-deficient mice (Mlh1−/−) colon crypts, which were previously analyzed using...
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Research: A prospective trial comparing programmable targeted long-read sequencing and short-read genome sequencing for genetic diagnosis of cerebellar ataxia
- Haloom Rafehi,
- Liam G. Fearnley,
- Justin Read,
- Penny Snell,
- Kayli C. Davies,
- Liam Scott,
- Greta Gillies,
- Genevieve C. Thompson,
- Tess A. Field,
- Aleena Eldo,
- Simon Bodek,
- Ernest Butler,
- Luke Chen,
- John Drago,
- Himanshu Goel,
- Anna Hackett,
- G. Michael Halmagyi,
- Andrew Hannaford,
- Katya Kotschet,
- Kishore R. Kumar,
- Smitha Kumble,
- Matthew Lee-Archer,
- Abhishek Malhotra,
- Mark Paine,
- Michael Poon,
- Kate Pope,
- Katrina Reardon,
- Steven Ring,
- Anne Ronan,
- Matthew Silsby,
- Renee Smyth,
- Chloe Stutterd,
- Mathew Wallis,
- John Waterston,
- Thomas Wellings,
- Kirsty West,
- Christine Wools,
- Kathy H.C. Wu,
- David J. Szmulewicz,
- Martin B. Delatycki,
- Melanie Bahlo,
- and Paul J. Lockhart
Genome Res. April 2025 35: 769-785; Published in Advance February 27, 2025, doi:10.1101/gr.279634.124
...LRS providers are Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). PacBio performs sequencing by synthesis and requires the construction of a library of circular DNA molecules. In contrast, ONT is a nanopore-based technology. The DNA sequence is determined by measuring nucleotide...
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Method: High-throughput single-cell functional elucidation of neurodevelopmental disease–associated genes reveals convergent mechanisms altering neuronal differentiation
- Matthew A. Lalli,
- Denis Avey,
- Joseph D. Dougherty,
- Jeffrey Milbrandt,
- and Robi D. Mitra
Genome Res. September 2020 30: 1317-1331; Published in Advance September 4, 2020, doi:10.1101/gr.262295.120
...protocols. In brief, HEK293T cells were seeded at a density of 1 million cells per well of a six-well plate and transfected with 2 μg of DNA comprising 1 µg gRNA-transfer plasmid, 750 ng psPAX2, and 250 ng pMD2.G. psPAX2 and pMD2.G were gifts from Didier Trono (Addgene 12259 and 12260). Cells were...
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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
...-free water ready for the ONT direct RNA sequencing protocol. Given the poorer quality of artificially polyadenylated reads, it might be beneficial to adopt a more gentle approach (Yan et al. 2018).PacBio cDNA sequencingPacBio SMRTbell libraries were prepared according to the manufacturer's instructions...

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