{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T08:41:51Z","timestamp":1789720911004,"version":"4.0.1"},"posted":{"date-parts":[[2025,6,27]]},"group-title":"Bioinformatics","reference-count":76,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T00:00:00Z","timestamp":1750982400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["1R01NS124916"],"award-info":[{"award-number":["1R01NS124916"]}],"id":[{"id":"https:\/\/ror.org\/00k86s890","id-type":"ROR","asserted-by":"publisher"}]},{"award":["1R24NS132738"],"award-info":[{"award-number":["1R24NS132738"]}],"id":[{"id":"https:\/\/ror.org\/00k86s890","id-type":"ROR","asserted-by":"publisher"}]},{"award":["ASAP-000301"],"award-info":[{"award-number":["ASAP-000301"]}],"id":[{"id":"https:\/\/ror.org\/03zj4c476","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,6,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Motivation<\/jats:title>\n                  <jats:p>Extracellular vesicle (EV) RNA sequencing has emerged as a powerful approach for studying RNA biomarkers and intercellular communication. Nevertheless, the extremely low abundance, fragmented nature and ubiquitous tissue origin of EV RNAs, alongside potential contamination from co-isolated materials, such as free DNA and bacterial RNA, pose substantial analytical challenges. These complexities highlight a pressing need for a standardized, computational workflow that ensures robust quality control and EV RNA characterization.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Here, we present EVscope, an open-source bioinformatics pipeline designed specifically for processing EV RNA-seq datasets. EVscope employs an optimized genome-wide expectation-maximization (EM) algorithm that significantly improves multi-mapping read assignment at single-base resolution by effectively leveraging alignment scores (AS) and local read coverage, specifically tailored for fragmented and low-abundance EV RNAs. Notably, EVscope uniquely generates EM-based BigWig files for downstream analysis, a capability currently unavailable in existing EM-based BigWig quantification tools. The pipeline systematically integrates 27 major steps, including quality control, analysis of library structure, contamination assessment, read alignment, read strandedness detection, UMI-based deduplication, RNA quantification, genomic DNA (gDNA) contamination correction, cellular and tissue source inference and visualization with a comprehensive HTML report. EVscope incorporates a comprehensive, updated annotation covering 19 distinct RNA biotypes, encompassing protein-coding genes, lncRNAs, miRNAs, piRNAs, retrotransposons (LINEs, SINEs, ERVs), and additional non-coding RNAs (tRNAs, rRNAs, snoRNAs). Furthermore, it leverages two highly balanced circRNA detection algorithms for robust circular RNA identification. Notably, a downstream module enables the inference of the tissue\/cellular origins of EV RNAs using bulk and single-cell RNA-seq reference datasets. EVscope is implemented as a convenient, single-command Bash pipeline leveraging Conda-managed standard software packages and custom scripts, ensuring reproducibility and straightforward deployment.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Availability and implementation<\/jats:title>\n                  <jats:p>\n                    Code, documentation, and tutorials are available at GitHub (\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/TheDongLab\/EVscope\">https:\/\/github.com\/TheDongLab\/EVscope<\/jats:ext-link>\n                    ) and archived on Zenodo (\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/zenodo.org\/records\/15577789\">https:\/\/zenodo.org\/records\/15577789<\/jats:ext-link>\n                    ).\n                  <\/jats:p>\n   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