{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:01Z","timestamp":1792790341458,"version":"4.5.0"},"posted":{"date-parts":[[2026,5,31]]},"group-title":"Bioinformatics","reference-count":31,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,31]],"date-time":"2026-05-31T00:00:00Z","timestamp":1780185600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["NCI R33 CA281919"],"award-info":[{"award-number":["NCI R33 CA281919"]}]},{"name":"National Institutes of Health","award":["NIGMS R35 GM142647"],"award-info":[{"award-number":["NIGMS R35 GM142647"]}]},{"name":"University of Virginia Cancer Center","award":["P30 CA44579"],"award-info":[{"award-number":["P30 CA44579"]}],"id":[{"id":"https:\/\/ror.org\/04w75nz84","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,17]]},"abstract":"<jats:title>Summary<\/jats:title>\n                <jats:p>Most human genes produce multiple RNA isoforms, yet it remains unclear which isoforms are translated into stable, functional proteins. Long-read RNA sequencing resolves full-length transcript structures and, when paired with mass spectrometry, can provide empirical evidence of isoform translation. Despite this opportunity, comprehensive workflows integrating isoform discovery, open reading frame prediction, peptide identification, and protein inference remain limited, leaving users to handle these steps piecemeal. Here, we present LRP2, a modular, end-to-end long-read proteogenomics pipeline built in Nextflow. LRP2 scales transcript discovery to hundreds of samples via PacBio\u2019s latest Isocall tool, removes technical artifacts with SQANTI QC, generates and classifies predicted proteomes via CPAT and SQANTI Protein, performs multi-group differential expression and usage analysis via edgeR, DRIMSeq, and a long-read adaptation of LeafCutter, and integrates protein-level evidence from DDA and DIA MS data through FragPipe. For cross-dataset comparison of novel isoforms, LRP2 employs deterministic splice-junction, coordinate-based isoform identifiers. Used as an integrated pipeline, LRP2 enables the detection of novel peptides and improves the protein isoform inference to confirm protein isoform translation.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Availability and implementation<\/jats:title>\n                  <jats:p>\n                    LRP2 is freely available as a modular Nextflow pipeline at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/sheynkman-lab\/LRP2\">https:\/\/github.com\/sheynkman-lab\/LRP2<\/jats:ext-link>\n                    (v2.0.0, archived at\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/doi.org\/10.5281\/zenodo.22795865\">https:\/\/doi.org\/10.5281\/zenodo.22795865<\/jats:ext-link>\n                    ). LRP2 supports Docker, Apptainer, and Conda environments with GENCODE references.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Contact<\/jats:title>\n                  <jats:p>\n                    Megan Schertzer,\n                    <jats:email>cwp5au@virginia.edu<\/jats:email>\n                  <\/jats:p>\n                  <jats:p>\n                    David Knowles,\n                    <jats:email>daknowles@nygenome.org<\/jats:email>\n                  <\/jats:p>\n                  <jats:p>\n                    Gloria Sheynkman,\n                    <jats:email>gs9yr@virginia.edu<\/jats:email>\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.05.27.728216","type":"posted-content","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T04:35:11Z","timestamp":1780288511000},"source":"Crossref","is-referenced-by-count":1,"title":["LRP2: A proteogenomics pipeline for long-read informed protein isoform analysis and discovery"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4997-8221","authenticated-orcid":false,"given":"Megan D.","family":"Schertzer","sequence":"first","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"},{"name":"New York Genome Center, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-1711-5248","authenticated-orcid":false,"given":"Julia T.","family":"Lewandowski","sequence":"additional","affiliation":[{"name":"New York Genome Center, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6018-4428","authenticated-orcid":false,"given":"Emily F.","family":"Watts","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0359-8410","authenticated-orcid":false,"given":"Will","family":"Rosenow","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9598-8100","authenticated-orcid":false,"given":"Madison M.","family":"Mehlferber","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"},{"name":"Department of Biochemistry and Molecular Genetics, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9511-7719","authenticated-orcid":false,"given":"Erin D.","family":"Jeffery","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4935-5721","authenticated-orcid":false,"given":"Scott I.","family":"Adamson","sequence":"additional","affiliation":[{"name":"New York Genome Center, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jocelyne","family":"Bruand","sequence":"additional","affiliation":[{"name":"Pacific Biosciences, Menlo Park, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1074-5095","authenticated-orcid":false,"given":"Elizabeth","family":"Tseng","sequence":"additional","affiliation":[{"name":"Pacific Biosciences, Menlo Park, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5840-652X","authenticated-orcid":false,"given":"Yaseswini","family":"Neelamraju","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4771-628X","authenticated-orcid":false,"given":"Francine E.","family":"Garrett-Bakelman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, UVA, Charlottesville, VA, USA"},{"name":"Department of Medicine, UVA, Charlottesville, VA, USA"},{"name":"UVA Comprehensive Cancer Center, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3296-0677","authenticated-orcid":false,"given":"Egor","family":"Dolzhenko","sequence":"additional","affiliation":[{"name":"Pacific Biosciences, Menlo Park, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7408-146X","authenticated-orcid":false,"given":"David A.","family":"Knowles","sequence":"additional","affiliation":[{"name":"New York Genome Center, New York, NY, USA"},{"name":"Systems Biology Department, Columbia University, New York, NY, USA"},{"name":"Computer Science Department, Columbia University, New York, NY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4223-9947","authenticated-orcid":false,"given":"Gloria","family":"Sheynkman","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biological Physics, UVA, Charlottesville, VA, USA"},{"name":"Department of Biochemistry and Molecular Genetics, UVA, Charlottesville, VA, USA"},{"name":"UVA Comprehensive Cancer Center, UVA, Charlottesville, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092107152474000_2026.05.27.728216v2.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.27"},{"key":"2026092107152474000_2026.05.27.728216v2.2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.0c00407"},{"key":"2026092107152474000_2026.05.27.728216v2.3","doi-asserted-by":"publisher","DOI":"10.64898\/2026.09.08.749180"},{"key":"2026092107152474000_2026.05.27.728216v2.4","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0132628"},{"key":"2026092107152474000_2026.05.27.728216v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/j.gene.2012.07.083"},{"issue":"5","key":"2026092107152474000_2026.05.27.728216v2.6","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1038\/nmeth.4256","article-title":"MSFragger: Ultrafast and Comprehensive Peptide Identification in Mass Spectrometry-Based Proteomics","volume":"14","year":"2017","journal-title":"Nature Methods"},{"key":"2026092107152474000_2026.05.27.728216v2.7","doi-asserted-by":"publisher","DOI":"10.64898\/2026.08.06.743133"},{"key":"2026092107152474000_2026.05.27.728216v2.8","doi-asserted-by":"publisher","DOI":"10.7554\/elife.108878.1"},{"key":"2026092107152474000_2026.05.27.728216v2.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41588-017-0004-9"},{"issue":"1","key":"2026092107152474000_2026.05.27.728216v2.10","doi-asserted-by":"crossref","first-page":"1228","DOI":"10.1080\/15476286.2022.2141938","article-title":"Characterization of Protein Isoform Diversity in Human Umbilical Vein Endothelial Cells via Long-Read Proteogenomics","volume":"19","year":"2022","journal-title":"RNA Biology"},{"key":"2026092107152474000_2026.05.27.728216v2.11","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-022-02624-y"},{"key":"2026092107152474000_2026.05.27.728216v2.12","first-page":"1","article-title":"Transcriptomics in the Era of Long-Read Sequencing","volume":"28","year":"2025","journal-title":"Nature Reviews. Genetics, March"},{"key":"2026092107152474000_2026.05.27.728216v2.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01369-0"},{"issue":"11","key":"2026092107152474000_2026.05.27.728216v2.14","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1038\/nmeth.3144","article-title":"Proteogenomics: Concepts, Applications and Computational Strategies","volume":"11","year":"2014","journal-title":"Nature Methods"},{"key":"2026092107152474000_2026.05.27.728216v2.15","doi-asserted-by":"publisher","DOI":"10.1038\/nature08909"},{"key":"2026092107152474000_2026.05.27.728216v2.16","doi-asserted-by":"crossref","first-page":"1356","DOI":"10.12688\/f1000research.8900.2","article-title":"DRIMSeq: A Dirichlet-Multinomial Framework for Multivariate Count Outcomes in Genomics","volume":"5","year":"2016","journal-title":"F1000Research"},{"key":"2026092107152474000_2026.05.27.728216v2.17","doi-asserted-by":"publisher","DOI":"10.1038\/ng.259"},{"issue":"5","key":"2026092107152474000_2026.05.27.728216v2.18","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/s41592-024-02229-2","article-title":"SQANTI3: Curation of Long-Read Transcriptomes for Accurate Identification of Known and Novel Isoforms","volume":"21","year":"2024","journal-title":"Nature Methods"},{"issue":"7","key":"2026092107152474000_2026.05.27.728216v2.19","doi-asserted-by":"crossref","first-page":"1349","DOI":"10.1038\/s41592-024-02298-3","article-title":"Systematic Assessment of Long-Read RNA-Seq Methods for Transcript Identification and Quantification","volume":"21","year":"2024","journal-title":"Nature Methods"},{"key":"2026092107152474000_2026.05.27.728216v2.20","doi-asserted-by":"publisher","DOI":"10.1101\/2023.05.15.540865"},{"issue":"1451024","key":"2026092107152474000_2026.05.27.728216v2.21","first-page":"1451024","article-title":"A Proteogenomic Atlas of the Human Neural Retina","volume":"15","year":"2024","journal-title":"Frontiers in Genetics"},{"key":"2026092107152474000_2026.05.27.728216v2.22","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp616"},{"key":"2026092107152474000_2026.05.27.728216v2.23","first-page":"1","article-title":"Perplexity as a Metric for Isoform Diversity in the Human Transcriptome","volume":"2","year":"2026","journal-title":"Genome Biology, September"},{"key":"2026092107152474000_2026.05.27.728216v2.24","doi-asserted-by":"publisher","DOI":"10.1038\/nrg.2015.3"},{"issue":"1","key":"2026092107152474000_2026.05.27.728216v2.25","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1146\/annurev-anchem-071015-041722","article-title":"Proteogenomics: Integrating next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation","volume":"9","year":"2016","journal-title":"Annual Review of Analytical Chemistry (Palo Alto, Calif"},{"key":"2026092107152474000_2026.05.27.728216v2.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-023-01714-x"},{"key":"2026092107152474000_2026.05.27.728216v2.27","doi-asserted-by":"publisher","DOI":"10.1038\/nature07509"},{"key":"2026092107152474000_2026.05.27.728216v2.28","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt006"},{"issue":"100077","key":"2026092107152474000_2026.05.27.728216v2.29","first-page":"100077","article-title":"IonQuant Enables Accurate and Sensitive Label-Free Quantification with FDR-Controlled Match-between-Runs","volume":"20","year":"2021","journal-title":"Molecular & Cellular Proteomics"},{"issue":"1","key":"2026092107152474000_2026.05.27.728216v2.30","doi-asserted-by":"crossref","first-page":"4154","DOI":"10.1038\/s41467-023-39869-5","article-title":"Analysis of DIA Proteomics Data Using MSFragger-DIA and FragPipe Computational Platform","volume":"14","year":"2023","journal-title":"Nature Communications"},{"key":"2026092107152474000_2026.05.27.728216v2.31","first-page":"1","article-title":"Identification of Non-Canonical Peptides with moPepGen","volume":"16","year":"2025","journal-title":"Nature Biotechnology"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.27.728216","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T14:16:27Z","timestamp":1790000187000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.27.728216"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,31]]},"references-count":31,"URL":"https:\/\/doi.org\/10.64898\/2026.05.27.728216","relation":{},"subject":[],"published":{"date-parts":[[2026,5,31]]},"subtype":"preprint"}}