Dehghannasiri Roozbeh, Kokot Marek, Starr Alexander L, Maziarz Jamie, Gordon Tal, Tan Serena Y, Wang Peter L, Voskoboynik Ayelet, Musser Jacob M, Deorowicz Sebastian, Salzman Julia
Department of Biomedical Data Science, Stanford University, Stanford, 94305, USA.
Department of Algorithmics and Software, v, Gliwice, Poland.
bioRxiv. 2024 Dec 24:2024.12.24.630263. doi: 10.1101/2024.12.24.630263.
Typical high-throughput single-cell RNA-sequencing (scRNA-seq) analyses are primarily conducted by (pseudo)alignment, through the lens of annotated gene models, and aimed at detecting differential gene expression. This misses diversity generated by other mechanisms that diversify the transcriptome such as splicing and V(D)J recombination, and is blind to sequences missing from imperfect reference genomes. Here, we present sc-SPLASH, a highly efficient pipeline that extends our SPLASH framework for statistics-first, reference-free discovery to barcoded scRNA-seq (10x Chromium) and spatial transcriptomics (10x Visium); we also provide its optimized module for preprocessing and -mer counting in barcoded data, BKC, as a standalone tool. sc-SPLASH rediscovers known biology including V(D)J recombination and cell-type-specific alternative splicing in human and trans-splicing in tunicate () and when applied to spatial datasets, detects sequence variation including tumor-specific somatic mutation. In sponge () and tunicate (), we uncover secreted repeat proteins expressed in immune-type cells and regulated during development; the sponge genes were absent from the reference assembly. sc-SPLASH provides a powerful alternative tool for exploring transcriptomes that is applicable to the breadth of life's diversity.
典型的高通量单细胞RNA测序（scRNA-seq）分析主要通过（伪）比对来进行，以注释基因模型为视角，旨在检测差异基因表达。这忽略了由其他使转录组多样化的机制（如剪接和V(D)J重组）产生的多样性，并且对不完美参考基因组中缺失的序列视而不见。在这里，我们展示了sc-SPLASH，这是一种高效的流程，它将我们用于基于统计的无参考发现的SPLASH框架扩展到条形码scRNA-seq（10x Chromium）和空间转录组学（10x Visium）；我们还提供了其在条形码数据中进行预处理和计数的优化模块BKC作为独立工具。sc-SPLASH重新发现了已知的生物学现象，包括人类中的V(D)J重组和细胞类型特异性可变剪接以及被囊动物中的反式剪接，并且当应用于空间数据集时，检测到包括肿瘤特异性体细胞突变在内的序列变异。在海绵动物和被囊动物中，我们发现了在免疫型细胞中表达并在发育过程中受到调控的分泌型重复蛋白；参考组装中没有海绵动物的基因。sc-SPLASH为探索转录组提供了一个强大的替代工具，适用于生命多样性的各个方面。
