Single-Molecule Sequencing of RNA
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Recent advances in RNA sequencing technologies have revolutionized the field of transcriptomics, shedding new light on the complexities of RNA biology. Single-molecule RNA sequencing, in particular, has opened unprecedented avenues for exploring gene expression, isoform diversity, RNA modifications, and dynamic transcript behaviors at single-molecule resolution. This transition from bulk RNA analyses to single-molecule studies represents a paradigm shift, enabling researchers to study the transcriptome with unparalleled accuracy and detail one molecule at a time.
With this primer, the authors provide an understandable and comprehensive introduction to single-molecule RNA sequencing, beginning with a historical perspective and progressing to the latest technological developments. Key topics include principles of the currently most relevant third-generation single-molecule platforms from PacBio and Oxford Nanopore Technologies, developments on RNA library preparation, sequencing workflows, data analysis pipelines, and examples of real-world applications. Throughout the chapters, the authors emphasize both the opportunities and challenges presented by single-molecule RNA-seq, from its technical limitations to its unique capabilities in resolving full-length transcripts, detecting rare isoforms, and mapping epitranscriptomic modifications.
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Dina Grohmann is fascinated by the heterogeneous nature of biology. Combining single-molecule biophysics, biochemistry, organismic and molecular microbiology, and state-of-the-art single-molecule sequencing, her research aims to gain a deeper understanding of the molecular machineries that are crucially involved in the cellular RNA metabolism—one molecule at a time. This includes research on transcriptional and posttranscriptional regulation in archaea and eukaryotes and the mammalian RNA silencing pathway. Dina Grohmann studied biology at the Heinrich Heine-University in Düsseldorf and did her Ph.D. at the Max Planck Institute for Molecular Physiology. She obtained her Ph.D. in Physical Biochemistry in 2006. In 2007, she joined the group of Prof. Finn Werner at University College London as a postdoctoral researcher, followed by a position as a junior research group leader at the Technical University of Braunschweig. In 2015, she was appointed as an associate professor at the University of Regensburg, followed by a full professorship in 2019 at the University of Regensburg.
Felix Grünberger is a postdoctoral researcher in the Microbiology Department and Archaea Centre at the University of Regensburg. He earned his Ph.D. in Biology in 2021, focusing on transcriptomics and gene regulation in archaea. His current research combines nanopore sequencing, bioinformatics, and molecular microbiology to investigate transcriptional regulation and stress responses in extremophiles. Felix’s recent work provides validated protocols for nanopore RNA-seq in prokaryotes, facilitating studies on transcriptional heterogeneity and RNA processing across diverse microbial systems.
