Identification and quantification of alternative polyadenylation sites in single cell RNA-seq data using scPAISO

Yongjie Liu; Peiwen Xiong; Songyang Li; Xinjia Liu; Tao Liu; Qinglan Yang; Shuting Wu; Hongyan Peng; Yana Li; Lingling Zhang; Yafei Deng; Yong Zhu; Junping Wang; Youcai Deng

doi:10.1016/j.jgg.2026.07.013

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Identification and quantification of alternative polyadenylation sites in single cell RNA-seq data using scPAISO

doi: 10.1016/j.jgg.2026.07.013

a. Pediatrics Research Institute of Hunan Province, the Affiliated Children's Hospital of Xiangya School of Medicine, Central South University (Hunan children's hospital), Changsha, Hunan 410007, China;
b. The School of Pediatrics, Hengyang Medical School, University of South China (Hunan Children's Hospital), Changsha, Hunan 410007, China;
c. Department of Pharmacology and Department of Clinical Hematology, College of Pharmacy and Laboratory Medicine Science, Army Medical University, Chongqing 400038, China;
d. State Key Laboratory of Trauma and Chemical Poisoning, Institute of Combined Injury, College of Preventive Medicine, Army Medical University, Chongqing 400038, China;
e. Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immune Medicine, Ministry of Education, Hefei, Anhui 230032, China;
f. College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China

Funds:

National Key Research and Development Project (2020YFA0113500)

and the Key Research and Development Program Project of the State Key Laboratory of Trauma and Chemical Poisoning (2024K004).

the Natural Science Foundation of Changsha, Hunan Province (kq2208087, China)

the Projects of Army Medical University (2022XJS06, 2023XQN20, 2023XQN19, China)

the Chongqing Municipal Education Commission Foundation (KJZD-M202412802, China)

Scientific Research Project of Hunan Provincial Health Commission (A202302077875, B202302078452, China)

This study was supported by funding from the National Natural Science Foundation of China (82273938, 82473935, 82473934, 32100616, 82304511)

Hunan Provincial Natural Science Foundation of China (2023JJ40341, 2023JJ40352)

Health Research Project of Hunan Provincial Health Commission (20256222)

Received Date: 2026-07-09
Accepted Date: 2026-07-31
Rev Recd Date: 2026-07-30

Available Online: 2026-08-08

Abstract

Abstract

Alternative polyadenylation (APA) generates transcript diversity by producing mRNA isoforms with distinct 3′ untranslated regions (3′ UTRs) or coding sequences. Existing single-cell RNA sequencing (scRNA-seq) methods for APA analysis primarily rely on Read2 data, which lacks precise cleavage site (CS) information and limits accurate polyadenylation site (PAS) identification. Here, we present single-cell PolyAdenylation ISOform quantification (scPAISO), a computational pipeline that leverages the often-discarded Read1 from 3′ tag-based scRNA-seq to enable de novo PAS identification and PAS isoform quantification. Unlike existing approaches, scPAISO directly captures mRNA 3′ end cleavage sites, resulting in stronger AAUAAA motif enrichment, sharper PAS peaks, and improved spatial resolution for resolving closely spaced PASs. Across multiple datasets and biological systems, scPAISO robustly identified PASs and quantified APA dynamics, revealing stage-specific 3′ UTR lengthening during hematopoietic differentiation, widespread 3′ UTR remodeling in systemic sclerosis, and tissue-specific polyadenylation preferences associated with distinct RNA-binding protein programs in mice. scPAISO provides an accurate and scalable framework for single-cell APA analysis, enabling high-resolution characterization of post-transcriptional regulation and transcriptome diversity in development, physiology, and disease.

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