TY - JOUR A1 - Hammer, Paul A1 - Banck, Michaela S. A1 - Amberg, Ronny A1 - Wang, Cheng A1 - Petznick, Gabriele A1 - Luo, Shujun A1 - Khrebtukova, Irina A1 - Schroth, Gary P. A1 - Beyerlein, Peter A1 - Beutler, Andreas S. T1 - mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain JF - Genome Research N2 - mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system. Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6446 SN - 1549-5469 VL - 20 IS - 6 ER - TY - JOUR A1 - Petrillo, Mauro A1 - Fabbri, Marco A1 - Kagkli, Dafni Maria A1 - Querci, Maddalena A1 - Van den Eede, Guy A1 - Alm, Erik A1 - Aytan-Aktug, Derya A1 - Capella-Gutierrez, Salvador A1 - Carrillo, Catherine A1 - Cestaro, Alessandro A1 - Chan, Kok-Gan A1 - Coque, Teresa A1 - Endrullat, Christoph A1 - Gut, Ivo A1 - Hammer, Paul A1 - Kay, Gemma L. A1 - Madec, Jean-Yves A1 - Mather, Alison E. A1 - McHardy, Alice Carolyn A1 - Naas, Thierry A1 - Paracchini, Valentina A1 - Peter, Silke A1 - Pightling, Arthur A1 - Raffael, Barbara A1 - Rossen, John A1 - Ruppé, Etienne A1 - Schlaberg, Robert A1 - Vanneste, Kevin A1 - Weber, Lukas M. A1 - Westh, Henrik A1 - Angers-Loustau, Alexandre T1 - A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing [version 1; peer review: 2 approved with reservations] JF - F1000Research N2 - Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain “live” (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines’ implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community. KW - antimicrobial resistance KW - bioinformatics KW - next-generation sequencing KW - benchmarking Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-15699 ER -