{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T11:12:05Z","timestamp":1790248325022,"version":"4.1.0"},"reference-count":139,"publisher":"OAE Publishing Inc.","issue":"3","content-domain":{"domain":["oaepublish.com"],"crossmark-restriction":false},"short-container-title":["Extracell Vesicles Circ Nucleic Acids."],"abstract":"<jats:p>Aim: Small RNA sequencing (small RNA-Seq) is a widely used and important method for biomarker discovery in extracellular vesicles (EVs), which carry small RNAs among their cargo and protect them from degradation in liquid biopsies. However, existing computational tools for small RNA-Seq data are not tailored to EV-RNA.<\/jats:p>\n                  <jats:p>Methods: We present comprehensive and bias-aware analysis of small RNA gene expression (caRNAge), a biomarker discovery pipeline for small RNA-Seq data intended for users without extensive bioinformatic expertise. Ready to use as a Snakemake pipeline, caRNAge assists users in bias-aware normalization, differential gene expression analysis, and pathway over-representation analysis.<\/jats:p>\n                  <jats:p>Results: Possible confounding factors can be identified and corrected to maximize data comparability between experiments and biomarker robustness. caRNAge is not limited to miRNAs; it also supports the detection of miRNA isomer (isomiRs), tRNA-derived fragments (tRFs), transfer RNA (tRNAs), and other small RNA species, thereby enhancing the specificity of biomarker signatures. Specific tissues can be selected for target prediction and pathway analysis to eliminate false-positive results. All results are provided in accessible and interactive HTML reports. We provide example data for each step of the pipeline to demonstrate caRNAge\u2019s capabilities and advantages across three EV-miRNA biomarker discovery use cases. The pipeline is not limited to EV-miRNA data but can be used for data evaluation in any small RNA-Seq experiment.<\/jats:p>\n                  <jats:p>Conclusion: By assisting users at every step of the discovery workflow, caRNAge fills an important gap in the field of EV-associated transcriptomic biomarker development. The pipeline is freely available at https:\/\/gitlab.lrz.de\/kirchner\/carnage.<\/jats:p>","DOI":"10.20517\/evcna.2026.78","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T10:36:04Z","timestamp":1790246164000},"page":"1554-81","update-policy":"https:\/\/doi.org\/10.20517\/oae.crossmark.policy","source":"Crossref","is-referenced-by-count":0,"title":["Comprehensive and bias-aware analysis of small RNA NGS data for biomarker detection with caRNAge"],"prefix":"10.20517","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3878-0148","authenticated-orcid":true,"given":"Benedikt","family":"Kirchner","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9356-7940","authenticated-orcid":true,"given":"Christian","family":"Gr\u00e4tz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johannes","family":"Kersting","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martina","family":"Schuster","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marlene","family":"Reithmair","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"List","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael W.","family":"Pfaffl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"8410","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb1596"},{"key":"B2","unstructured":"National Library of Medicine (US). Available from https:\/\/pubmed.ncbi.nlm.nih.gov\/. [accessed 23 September 2026]"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-cellbio-101512-122326"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12140"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12207"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201211138"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1007\/s00109-022-02189-0"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2021.662620"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.3390\/ijms23073692"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.gendis.2025.101682"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku656"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagrm.2019.03.005"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2021.668648"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-025-21561-x"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1186\/s13287-015-0116-z"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2014.08.027"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2019.11.078"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-030-86258-9_5"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.20517\/evcna.2025.68"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.3390\/ijms262412115"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbz124"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbs046"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-11-94"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac064"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-015-1308-8"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1910"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2019.00017"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1093\/nargab\/lqaa078"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1016\/bs.coac.2018.06.001"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1155\/2013\/951570"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv023"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1080\/15476286.2017.1320469"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1002\/bies.201800169"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1038\/nature03315"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2009.01.002"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2021.167070"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1093\/nargab\/lqab068"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx999"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.3390\/ncrna10010006"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky330"},{"key":"B41","unstructured":"nf-core\/smrnaseq (2026). Version 2.4.1. Available from https:\/\/nf-co.re\/smrnaseq\/2.2.4\/. [accessed 23 September 2026]"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.14806\/ej.17.1.200"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2009-10-3-r25"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1038\/srep41184"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab610"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2024.1388769"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts480"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.12688\/f1000research.29032.1"},{"key":"B49","unstructured":"FastQC: A quality control tool for high throughput sequence data (2010). Available from https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/. [accessed 23 September 2026]"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky1141"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt1181"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1038"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.1101\/2021.09.21.461238"},{"key":"B54","unstructured":"R Core Team. R: a language and environment for statistical computing (2024). Version 4.4.1. Available from https:\/\/www.r-project.org\/. [accessed 23 September 2026]"},{"key":"B55","unstructured":"renv: Project Environments (2026). Available from https:\/\/cran.r-project.org\/web\/packages\/renv\/index.html. [accessed 23 September 2026]"},{"key":"B56","unstructured":"fastqcr: quality control of sequencing data (2023). Available from https:\/\/cran.r-project.org\/web\/packages\/fastqcr\/index.html. [accessed 23 September 2026]"},{"key":"B57","unstructured":"data.table: Extension of \u2018data.frame\u2019 (2026). Available from https:\/\/cran.r-project.org\/web\/packages\/data.table\/index.html. [accessed 23 September 2026]"},{"key":"B58","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-014-0550-8"},{"key":"B59","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv007"},{"key":"B60","unstructured":"EnhancedVolcano: publication-ready volcano plots with enhanced colouring and labeling (2018). Available from https:\/\/github.com\/kevinblighe\/EnhancedVolcano. [accessed 23 September 2026]"},{"key":"B61","unstructured":"DT: a wrapper of the JavaScript library \u2018DataTables\u2019 (2025). Available from https:\/\/cran.r-project.org\/web\/packages\/DT\/index.html. [accessed 23 September 2026]"},{"key":"B62","doi-asserted-by":"publisher","DOI":"10.1109\/tvcg.2014.2346248"},{"key":"B63","unstructured":"ComplexUpset (2020). Version 1.3.3. Available from https:\/\/zenodo.org\/records\/7314197. [accessed 23 September 2026]"},{"key":"B64","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1005752"},{"key":"B65","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa309"},{"key":"B66","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btac172"},{"key":"B67","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku631"},{"key":"B68","doi-asserted-by":"publisher","DOI":"10.7554\/elife.05005"},{"key":"B69","doi-asserted-by":"publisher","DOI":"10.1126\/science.1260419"},{"key":"B70","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaz1776"},{"key":"B71","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw377"},{"key":"B72","doi-asserted-by":"publisher","DOI":"10.1201\/9781138359444"},{"key":"B73","doi-asserted-by":"publisher","DOI":"10.1201\/9781315373461-1"},{"key":"B74","doi-asserted-by":"publisher","DOI":"10.1201\/9780429447273"},{"key":"B75","unstructured":"htmlwidgets: HTML Widgets for R (2023). Available from https:\/\/cran.r-project.org\/web\/packages\/htmlwidgets\/index.html. [accessed 23 September 2026]"},{"key":"B76","unstructured":"ggplot2: elegant graphics for data analysis. 2nd ed. Use R! Cham: Springer; 2016. Available from https:\/\/link.springer.com\/book\/10.1007\/978-3-319-24277-4. [accessed 23 September 2026]"},{"key":"B77","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2018.1481321"},{"key":"B78","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-31160-3"},{"key":"B79","unstructured":"Analyzing RNA-seq data with DESeq2 (2025). Available from https:\/\/bioconductor.org\/packages\/release\/bioc\/vignettes\/DESeq2\/inst\/doc\/DESeq2.html. [accessed 23 September 2026]"},{"key":"B80","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad392"},{"key":"B81","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz1022"},{"key":"B82","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.1.27"},{"key":"B83","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae909"},{"key":"B84","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw1092"},{"key":"B85","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad1025"},{"key":"B86","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad960"},{"key":"B87","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1072"},{"key":"B88","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkq1107"},{"key":"B89","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz896"},{"key":"B90","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0206239"},{"key":"B91","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/iyad031"},{"key":"B92","doi-asserted-by":"publisher","DOI":"10.1038\/75556"},{"key":"B93","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0047786"},{"key":"B94","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkh023"},{"key":"B95","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz969"},{"key":"B96","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkj025"},{"key":"B97","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae872"},{"key":"B98","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1094"},{"key":"B99","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-120213-092023"},{"key":"B100","doi-asserted-by":"publisher","DOI":"10.1093\/gigascience\/giac083"},{"key":"B101","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1036"},{"key":"B102","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac884"},{"key":"B103","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa707"},{"key":"B104","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts698"},{"key":"B105","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav1741"},{"key":"B106","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-14-128"},{"key":"B107","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.90"},{"key":"B108","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz1021"},{"key":"B109","unstructured":"McKusick-Nathans Institute of Genetic Medicine - Johns Hopkins University (Baltimore MD). Online Mendelian Inheritance in Man, OMIM. Available from https:\/\/omim.org\/. [accessed 23 September 2026]"},{"key":"B110","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad1005"},{"key":"B111","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-016-0881-8"},{"key":"B112","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2931"},{"key":"B113","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2010-11-10-r106"},{"key":"B114","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2825"},{"key":"B115","doi-asserted-by":"publisher","DOI":"10.3390\/cancers15133446"},{"key":"B116","doi-asserted-by":"publisher","DOI":"10.1093\/biostatistics\/kxj037"},{"key":"B117","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.70005"},{"key":"B118","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2024.168571"},{"key":"B119","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-014-0078-0"},{"key":"B120","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2011.06.022"},{"key":"B121","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.02.053"},{"key":"B122","doi-asserted-by":"publisher","DOI":"10.3390\/ijms25020890"},{"key":"B123","doi-asserted-by":"publisher","DOI":"10.1038\/s41523-020-00211-7"},{"key":"B124","doi-asserted-by":"publisher","DOI":"10.1200\/jco.2005.02.8712"},{"key":"B125","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pmed.0020124"},{"key":"B126","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-025-03549-y"},{"key":"B127","doi-asserted-by":"publisher","DOI":"10.1016\/j.mam.2024.101269"},{"key":"B128","doi-asserted-by":"publisher","DOI":"10.3390\/life12030386"},{"key":"B129","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-9833-3_3"},{"key":"B130","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkw545"},{"key":"B131","doi-asserted-by":"publisher","DOI":"10.1101\/gr.205146.116"},{"key":"B132","doi-asserted-by":"publisher","DOI":"10.2174\/156652411794859250"},{"key":"B133","doi-asserted-by":"publisher","DOI":"10.1038\/nature08170"},{"key":"B134","doi-asserted-by":"publisher","DOI":"10.1186\/s13045-014-0078-0"},{"key":"B135","doi-asserted-by":"publisher","DOI":"10.1016\/j.ncrna.2021.10.002"},{"key":"B136","doi-asserted-by":"publisher","DOI":"10.1038\/s12276-022-00821-0"},{"key":"B137","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-022-00449-w"},{"key":"B138","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.27041"},{"key":"B139","doi-asserted-by":"publisher","DOI":"10.1093\/biostatistics\/kxv027"}],"container-title":["Extracellular Vesicles and Circulating Nucleic Acids"],"original-title":[],"link":[{"URL":"https:\/\/www.oaepublish.com\/articles\/evcna.2026.78","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.oaepublish.com\/articles\/evcna.2026.78","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T10:36:11Z","timestamp":1790246171000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.oaepublish.com\/articles\/evcna.2026.78"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":139,"journal-issue":{"issue":"3"},"URL":"https:\/\/doi.org\/10.20517\/evcna.2026.78","relation":{},"ISSN":["2767-6641"],"issn-type":[{"value":"2767-6641","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,24]]}}}