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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
...challenging to optimize, direct RNA sequencing allows for the QC of packaged lentiviral RNA and facilitates the development of improved vectors.DiscussionAchieving reliable production of intact LVs will have a significant impact on their manufacturing and clinical implementation. To this end, we have...
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Perspective: Challenges in identifying mRNA transcript starts and ends from long-read sequencing data
- Ezequiel Calvo-Roitberg,
- Rachel F. Daniels,
- and Athma A. Pai
Genome Res. November 2024 34: 1719-1734; doi:10.1101/gr.279559.124
...DNA molecules. The largest shift in length, however, is likely introduced by polymerase chain reaction (PCR) steps at the end of library preparation, which favors the amplification of shorter fragments (Fig. 2A; Shagin et al. 1999; Parekh et al. 2016). This bias should again equally affect misrepresentation...
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Resource: Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions
- Rodrigo P. Baptista,
- Yiran Li,
- Adam Sateriale,
- Mandy J. Sanders,
- Karen L. Brooks,
- Alan Tracey,
- Brendan R.E. Ansell,
- Aaron R. Jex,
- Garrett W. Cooper,
- Ethan D. Smith,
- Rui Xiao,
- Jennifer E. Dumaine,
- Peter Georgeson,
- Bernard J. Pope,
- Matthew Berriman,
- Boris Striepen,
- James A. Cotton,
- and Jessica C. Kissinger
Genome Res. January 2022 32: 203-213; Published in Advance November 11, 2021, doi:10.1101/gr.275325.121
...analysesStructural variation sites were calculated using NucDiff v2.0.3 (Khelik et al. 2017), and the major inversions observed between CpIRef and CpIA were verified by PCR. Primers to test both ends of each orientation were designed using Primer3 v0.4.0 (Supplemental Table S4; Untergasser et al. 2012...
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ARTICLE: Rapid and reliable cloning of PCR products.
- J B Lorens
Genome Res. November 1991 1: 140-141; doi:10.1101/gr.1.2.140
..., University of Bergen, N-5020 Bergen, Norway The cloning of PCR products is often desirable, especially when characteriz- ing heterogenous product populations. 1o achieve this, restriction sites are fre- quently included at the 5 ' end of each primer to allow direct cloning. How- ever, low cloning frequencies...
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Method: Theoretical framework for the difference of two negative binomial distributions and its application in comparative analysis of sequencing data
- Alicia Petrany,
- Ruoyu Chen,
- Shaoqiang Zhang,
- and Yong Chen
Genome Res. October 2024 34: 1636-1650; Published in Advance October 15, 2024, doi:10.1101/gr.278843.123
...embryonic fibroblasts (Islam et al. 2011). We used a gold-standard gene set, which contains DEGs validated by PCR experiments (Moliner et al. 2008; Kharchenko et al. 2014), to evaluate the sensitivity of each tool. DEGage displays strong performance by identifying 6626 genes with an FDR < 0...
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Method: NanoPARE: parallel analysis of RNA 5′ ends from low-input RNA
- Michael A. Schon,
- Max J. Kellner,
- Alexandra Plotnikova,
- Falko Hofmann,
- and Michael D. Nodine
Genome Res. December 2018 28: 1931-1942; Published in Advance October 24, 2018, doi:10.1101/gr.239202.118
....PARE methods capture sRNA cleavage products through a series of adapter-RNA ligations, Type IIS restriction enzyme digestions, and PCR amplifications (Addo-Quaye et al. 2008; German et al. 2008; Gregory et al. 2008). Because EndCut utilizes empirically determined models based on randomized sRNAs, it can...
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Method: Detection and characterization of jagged ends of double-stranded DNA in plasma
- Peiyong Jiang,
- Tingting Xie,
- Spencer C. Ding,
- Ze Zhou,
- Suk Hang Cheng,
- Rebecca W.Y. Chan,
- Wing-Shan Lee,
- Wenlei Peng,
- John Wong,
- Vincent W.S. Wong,
- Henry L.Y. Chan,
- Stephen L. Chan,
- Liona C.Y. Poon,
- Tak Y. Leung,
- K.C. Allen Chan,
- Rossa W.K. Chiu,
- and Y.M. Dennis Lo
Genome Res. August 2020 30: 1144-1153; Published in Advance August 14, 2020, doi:10.1101/gr.261396.120
...to bisulfite treatment and PCR amplification. If a molecule contained jagged ends with a 5′ protruding single strand, the methylation levels at CG sites proximal to the 3′ end (e.g., read2) would be lower than that close to the 5′ end (e.g., read1). Filled lollipops represent methylated Cs, and unfilled...
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Method: Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR
- Marta Puig,
- Jon Lerga-Jaso,
- Carla Giner-Delgado,
- Sarai Pacheco,
- David Izquierdo,
- Alejandra Delprat,
- Magdalena Gayà-Vidal,
- Jack F. Regan,
- George Karlin-Neumann,
- and Mario Cáceres
Genome Res. May 2020 30: 724-735; Published in Advance May 18, 2020, doi:10.1101/gr.255273.119
...-Soler et al. 2018), among other applications. Recently, ddPCR-based linkage calculations have also been used to phase variants separated by up to 200 kb (Regan et al. 2015), including deletions (Boettger et al. 2016), or for fusion transcript detection (Hoff et al. 2016). Here, we have developed new ddPCR...
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Research: Deficiency of nucleotide excision repair is associated with mutational signature observed in cancer
- Myrthe Jager,
- Francis Blokzijl,
- Ewart Kuijk,
- Johanna Bertl,
- Maria Vougioukalaki,
- Roel Janssen,
- Nicolle Besselink,
- Sander Boymans,
- Joep de Ligt,
- Jakob Skou Pedersen,
- Jan Hoeijmakers,
- Joris Pothof,
- Ruben van Boxtel,
- and Edwin Cuppen
Genome Res. July 2019 29: 1067-1077; Published in Advance June 20, 2019, doi:10.1101/gr.246223.118
...Institute, 3584 CT Utrecht, The Netherlands Corresponding author: ecuppen@umcutrecht.nlAbstractNucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging...
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Research: Distinct roles for RSC and SWI/SNF chromatin remodelers in genomic excision repair
- Kaitlynne A. Bohm,
- Amelia J. Hodges,
- Wioletta Czaja,
- Kathiresan Selvam,
- Michael J. Smerdon,
- Peng Mao,
- and John J. Wyrick
Genome Res. June 2021 31: 1047-1059; Published in Advance May 17, 2021, doi:10.1101/gr.274373.120
...), despite its broad effect on NER activity. Moreover, RT-qPCR analysis of the TFB1 gene, which is the yeast homolog of the gene encoding the p62 subunit (i.e., GTF2H1), shows no change in expression in the rsc2Δ mutant (Supplemental Fig. S12), consistent with published expression data (Hu et al. 2007...

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