{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T02:41:17Z","timestamp":1792464077160,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,28]]},"group-title":"Bioinformatics","reference-count":34,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T00:00:00Z","timestamp":1790553600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,28]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Alternative splicing is cell-type-specific and disease-relevant, but single-cell RNA sequencing is optimized for gene-level quantification, and how sequencing depth governs splice-junction recovery in the dominant 3\u2019-biased chemistries has not been quantified, so experiments cannot be designed to a depth target and datasets cannot be interpreted against expected recovery. We generated deep single-cell RNA sequencing of human airway epithelial cells (12 samples, 102 to 803 million uniquely mapped reads per sample) and benchmarked junction recovery in five annotated samples against matched bulk RNA sequencing from a 190-donor cohort. Junction detection did not saturate with depth, and validity was governed by read support rather than annotation status. Recovering 90% of the observed full-depth junction complement required approximately 52,250 reads per cell, several-fold more than gene-level analysis. Quantifying splicing was more depth-limited than detecting it, and rare cell types were limited by cell number rather than depth. Against matched bulk, single-cell recall plateaued at a practical ceiling set by sample size and sparse sampling, and was independent of distance from the 3\u2019 end. These results establish a framework for designing splicing experiments in deep 3\u2019-biased single-cell RNA sequencing, and provide an airway-epithelial dataset with matched bulk for benchmarking splicing methods.<\/jats:p>","DOI":"10.64898\/2026.09.22.753522","type":"posted-content","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T15:35:18Z","timestamp":1790609718000},"source":"Crossref","is-referenced-by-count":0,"title":["A framework for designing splice-junction experiments in deep 3\u2019 single-cell RNA sequencing"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6399-0249","authenticated-orcid":false,"given":"Aabida","family":"Saferali","sequence":"first","affiliation":[{"name":"Channing Division of Network Medicine, Brigham and Women\u2019s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Congjian","family":"Liu","sequence":"additional","affiliation":[{"name":"Division of Pulmonary and Critical Care Medicine, Mass General Brigham, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea S","family":"Vendrame","sequence":"additional","affiliation":[{"name":"Division of Pulmonary and Critical Care Medicine, Mass General Brigham, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kamakshi","family":"Bankoti","sequence":"additional","affiliation":[{"name":"Division of Pulmonary and Critical Care Medicine, Mass General Brigham, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose Santos","family":"Cabrera","sequence":"additional","affiliation":[{"name":"Channing Division of Network Medicine, Brigham and Women\u2019s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3997-5839","authenticated-orcid":false,"given":"Yohannes","family":"Tesfaigzi","sequence":"additional","affiliation":[{"name":"Division of Pulmonary and Critical Care Medicine, Mass General Brigham, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter J.","family":"Castaldi","sequence":"additional","affiliation":[{"name":"Channing Division of Network Medicine, Brigham and Women\u2019s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026101919311303000_2026.09.22.753522v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nature07509"},{"key":"2026101919311303000_2026.09.22.753522v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/ng.259"},{"key":"2026101919311303000_2026.09.22.753522v1.3","doi-asserted-by":"crossref","unstructured":"Saferali, A. , et al., Characterisation of a COPD-associated nephronectin (NPNT) functional splicing genetic variant in human lung tissue via long-read sequencing. 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