Single-cell alternative splicing analysis with Expedition reveals splicing dynamics during neuron differentiation

Alternative splicing (AS) generates isoform diversity critical for cellular identity and homeostasis, yet characterization of this diversity in single cells remains limited. We developed Expedition, a computational framework to categorize and visualize the heterogeneity of AS from single-cell transcriptomes. Expedition consists of (i) outrigger, a de novo splice graph transversal algorithm to detect AS from single cell RNA-seq; (ii) anchor, a Bayesian approach to assign splicing modalities and (iii) bonvoyage, using non-negative matrix factorization to visualize modality changes. By applying Expedition to single iPSCs undergoing neuron differentiation, we discover that 25% of AS exons exhibit bimodality and are flanked by longer and more conserved introns harboring distinct cis-regulatory motifs. Bimodal exons are highly dynamic during cellular transitions, preserve translatability, enriched in recently emerged genes and have conserved AS in mammals. Applying Expedition (http://github.com/YeoLab/Expedition) in single cells redefines our estimates and understanding of AS in evolution and biology. Overall design: Analysis of alternative splicing changes across motor neuron differentiation

可变剪接（Alternative Splicing, AS）可产生转录本多样性，这对维持细胞身份与稳态平衡至关重要，但目前对单细胞层面的该类多样性的表征仍十分有限。我们开发了Expedition这一计算框架，用于对单细胞转录组中的可变剪接异质性进行分类与可视化。该框架包含三大模块：(i) outrigger：一款可从单细胞RNA测序数据中检测可变剪接的从头剪接图遍历算法；(ii) anchor：一种用于分配剪接模式的贝叶斯方法；(iii) bonvoyage：采用非负矩阵分解来可视化剪接模式变化的工具。我们将Expedition应用于经历神经元分化的诱导多能干细胞（induced pluripotent stem cells, iPSCs）后发现，25%的可变剪接外显子呈现双峰性，其两侧侧翼为更长且更保守的内含子，且这些内含子携带独特的顺式调控基序。双峰性外显子在细胞转换过程中具有高度动态性，可维持翻译可行性，在新近演化出的基因中富集，并在哺乳动物中存在保守的可变剪接现象。将Expedition（http://github.com/YeoLab/Expedition）应用于单细胞研究，重新定义了我们对演化与生物学领域中可变剪接的评估与认知。整体设计：对运动神经元分化全过程中的可变剪接变化进行分析。