https://genome.cshlp.org/
april 25 issue
In this Part 2 of the Special Issue on Long-read DNA Sequencing Applications in Biology and Medicine, Guest Edited by Drs. Ana Conesa, Alexander Hoischen, and Fritz Sedlazeck, Genome Research publishes a diverse collection of research and methods highlighting novel applications and developments in long-read sequencing.
Papers in this issue focus on original research offering novel biological and clinical insights gained using long-read DNA and RNA sequencing technologies and other long molecule approaches. Reviews and Perspectives include a Hitchhiker’s Guide to long-read genomic analysis, and insights into the additional diagnostic yield of long-read sequencing in undiagnosed rare diseases, the complexity of cancer, the challenges in genome annotation, population genomics, transcriptomics, and epigenomics.
The issue also offers significant advances in long-read sequencing analysis, including novel methods for genome assembly and annotation, prioritization of structural variants in rare disease, characterization of complex repeats, RNA modification, mRNA and protein isoform diversity, tumor cell-free DNA profiling, and bacterial surveillance. Several of the studies are highlighted below.
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Unraveling undiagnosed rare disease cases by HiFi long-read genome sequencing
Wouter Steyaert, Lydia Sagath, German Demidov, Vicente A. Yépez, Anna Esteve-Codina, Julien Gagneur, Kornelia Ellwanger, Ronny Derks, Marjan Weiss, Amber den Ouden, Simone van den Heuvel, Hilde Swinkels, Nick Zomer, Marloes Steehouwer, Luke O’Gorman, Galuh Astuti, Kornelia Neveling, Rebecca Schüle, Jishu Xu, Matthis Synofzik, Danique Beijer, Holger Hengel, Ludger Schöls, Kristl G. Claeys, Jonathan Baets, Liedewei Van de Vondel, Alessandra Ferlini, Rita Selvatici, Heba Morsy, Marwa Saeed Abd Elmaksoud, Volker Straub, Juliane Müller, Veronica Pini, Luke Perry, Anna Sarkozy, Irina Zaharieva, Francesco Muntoni, Enrico Bugiardini, Kiran Polavarapu, Rita Horvath, Evan Reid, Hanns Lochmüller, Marco Spinazzi, Marco Savarese, Solve-RD DITF-ITHACA, Solve-RD DITF-Euro-NMD, Solve-RD DITF-RND, Solve-RD DITF-EpiCARE, Leslie Matalonga, Steven Laurie, Han G. Brunner, Holm Graessner, Sergi Beltran, Stephan Ossowski, Lisenka E.L.M. Vissers, Christian Gilissen, Alexander Hoischen, on behalf of the Solve-RD consortium
Steyaert and colleagues, as part of the European Solve-RD consortium effort, applied long-read genome sequencing to almost 300 individuals from previously undiagnosed rare disease families. They found (likely) disease-causing genetic variants in about 12% of previously unsolved families and additional candidate disease-causing structural variants in another 5.4% of these families, demonstrating the potential added value of long-read genome sequencing in undiagnosed rare diseases.
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Expanded methylome and quantitative trait loci detection by long-read profiling of personal DNA
Cristian Groza, Bing Ge, Warren A. Cheung, Tomi Pastinen, and Guillaume Bourque
Groza and colleagues use a pangenomic approach to identify structural variants (SVs) in almost 800 de novo assemblies obtained from Genomic Answers for Kids, use methylomes to genotype millions of non-reference CpGs within SVs (SV-CpGs), characterize their population frequency and methylation status, and associate tens-of-thousands of methylation quantitative-trait loci associated with SVs (SV-mQTLs). This study underscores the importance of assembly-based analyses of human traits.
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Multiple paralogs and recombination mechanisms contribute to the high incidence of 22q11.2 Deletion Syndrome
Lisanne Vervoort, Nicolas Dierckxsens, Marta Sousa Santos, Senne Meynants, Erika Souche, Ruben Cools, Tracy Heung, Koen Devriendt, Hilde Peeters, Donna McDonald-McGinn, Ann Swillen, Jeroen Breckpot, Beverly S. Emanuel, Hilde Van Esch, Anne S. Bassett, and Joris R. Vermeesch
Vervoort and colleagues combined fiber-FISH optical mapping with whole genome (ultra-)long read sequencing or rearrangement-specific long-range PCR on 22q11.2 deletion syndrome patients. They demonstrate that different paralogous segmental duplicon and palindromic AT-rich repeats are driving 22q11.2 rearrangements, as well as different inversion polymorphisms preceding rearrangement, and somatic mosaicism. This study resolves complex segmental duplicon structure to provide direct confirmation of the hypothesis that the rearrangements are caused by nonallelic homologous recombination between the low copy repeats on Chromosome 22 and provides a likely explanation for the high 22q11.2DS incidence.
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Multisample motif discovery and visualization for tandem repeats
Yaran Zhang, Marc Hulsman, Alex Salazar, Niccolò Tesi, Lydian Knoop, Sven van der Lee, Sanduni Wijesekera, Jana Krizova, Erik-Jan Kamsteeg, and Henne Holstege
Zhang and colleagues present MotifScope, a novel algorithm for characterization and visualization of tandem repeats. Comparison to established tools shows that MotifScope identifies a greater number of motifs and more accurately represents the underlying repeat sequence. MotifScope may be applied across diverse fields, including population genetics, clinical settings, and forensic analyses.
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Evaluation of strategies for evidence-driven genome annotation using long-read RNA-seq
Alejandro Paniagua, Cristina Agustin-García, Francisco J. Pardo-Palacios, Thomas Brown, Maite De Maria, Nancy D. Denslow, Camila J. Mazzoni, and Ana Conesa
Paniagua and colleagues analyze how long-read RNA sequencing platforms enhance genome annotation and incorporate long-read transcripts into an annotation pipeline applied to a nonmodel species, the Florida manatee. While annotations were highly concordant when compared to existing short-read-based annotation, they identified many novel loci represented by isofroms matching known sequences in other mammalian species. These results underscore the importance of using high-quality curated transcript models in combination with ab initio methods for effective genome annotation.
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Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications
Dayea Park and Can Cenik
Park and Cenik use nanopore direct RNA sequencing to detect allele-specific m6A RNA modifications (ASM) in both human and mouse cells. They find ASM sites with no nearby genetic differences suggesting that m6A modification may depend on factors beyond local sequence context. They also find that nucleotide identity of the positions surrounding the canonical DRACH motif may influence m6A deposition. Nanopore direct RNA sequencing to identify allele-specific m6A modification promises to advance our understanding of the role of genetics in determining mRNA modifications.
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De novo detection of somatic variants in high-quality long-read single-cell RNA sequencing data
Arthur Dondi, Nico Borgsmüller, Pedro F. Ferreira, Brian J. Haas, Francis Jacob, Viola Heinzelmann-Schwarz, Tumor Profiler Consortium, and Niko Beerenwinkel
Dondi and colleagues present LongSom, a computational workflow to call de novo somatic single-nucleotide variants (SNVs), copy-number alterations (CNAs), and gene fusions in long-read single-cell RNA-seq data. They apply LongSom to ovarian cancer samples and detect clinically relevant somatic SNVs that could not be detected with short-read single-cell RNA-seq and also found subclones with different predicted treatment outcomes. This advance enables the study of cancer evolution, tumor clonal heterogeneity, and treatment resistance.
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Rearrangements of viral and human genomes at human papillomavirus integration events and their allele-specific impacts on cancer genome regulation
Vanessa L. Porter, Michelle Ng, Kieran O’Neill, Signe MacLennan, Richard D. Corbett, Luka Culibrk, Zeid Hamadeh, Marissa Iden, Rachel Schmidt, Shirng-Wern Tsaih, Carolyn Nakisige, Martin Origa, Jackson Orem, Glenn Chang, Jeremy Fan, Ka Ming Nip, Vahid Akbari, Simon K. Chan, James Hopkins, Richard A. Moore, Eric Chuah, Karen L. Mungall, Andrew J. Mungall, Inanc Birol, Steven J.M. Jones, Janet S. Rader, and Marco A. Marra
Porter and colleagues performed long-read sequencing on over 70 cervical cancer genomes from a data set that was previously characterized using short-read sequencing and found recurrent structural rearrangement patterns at human papillomavirus (HPV) integration events. They characterized the genomic structures, methylation patterns, and transcriptional regulation associated with these loci providing insight into the role of integrated HPV in cervical cancer.
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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
Chen and colleagues present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which uses RCA and consensus calling based on genome-wide long-read nanopore sequencing to detect tumor fractions in cell-free DNA as low as 0.24%. This novel approach provides the opportunity for cost-effective and rapid tumor cell-free DNA profiling, addressing clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.
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