Fisheries College, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Jimei University, Xiamen, China.
Fourth Institute of Oceanography, Ministry of Natural Resources, Beihai, China.
Front Immunol. 2021 Sep 27;12:737332. doi: 10.3389/fimmu.2021.737332. eCollection 2021.
Fish is considered as a supreme model for clarifying the evolution and regulatory mechanism of vertebrate immunity. However, the knowledge of distinct immune cell populations in fish is still limited, and further development of techniques advancing the identification of fish immune cell populations and their functions are required. Single cell RNA-seq (scRNA-seq) has provided a new approach for effective in-depth identification and characterization of cell subpopulations. Current approaches for scRNA-seq data analysis usually rely on comparison with a reference genome and hence are not suited for samples without any reference genome, which is currently very common in fish research. Here, we present an alternative, i.e. scRNA-seq data analysis with a full-length transcriptome as a reference, and evaluate this approach on samples from -a teleost without any published genome. We show that it reconstructs well most of the present transcripts in the scRNA-seq data achieving a sensitivity equivalent to approaches relying on genome alignments of related species. Based on cell heterogeneity and known markers, we characterized four cell types: T cells, B cells, monocytes/macrophages (Mo/MΦ) and NCC (non-specific cytotoxic cells). Further analysis indicated the presence of two subsets of Mo/MΦ including M1 and M2 type, as well as four subsets in B cells, i.e. mature B cells, immature B cells, pre B cells and early-pre B cells. Our research will provide new clues for understanding biological characteristics, development and function of immune cell populations of teleost. Furthermore, our approach provides a reliable alternative for scRNA-seq data analysis in teleost for which no reference genome is currently available.
鱼类被认为是阐明脊椎动物免疫进化和调控机制的最佳模型。然而，鱼类中不同免疫细胞群体的知识仍然有限，需要进一步开发技术来鉴定鱼类免疫细胞群体及其功能。单细胞 RNA 测序（scRNA-seq）为有效深入鉴定和描述细胞亚群提供了新方法。目前的 scRNA-seq 数据分析方法通常依赖于与参考基因组的比较，因此不适合没有任何参考基因组的样本，而这在鱼类研究中目前非常常见。在这里，我们提出了一种替代方法，即使用全长转录组作为参考的 scRNA-seq 数据分析，并在没有发表的基因组的硬骨鱼类样本上评估了该方法。我们表明，它可以很好地重建 scRNA-seq 数据中的大多数现有转录本，其灵敏度相当于依赖于相关物种基因组比对的方法。基于细胞异质性和已知标记，我们鉴定了四种细胞类型：T 细胞、B 细胞、单核细胞/巨噬细胞（Mo/MΦ）和 NCC（非特异性细胞毒性细胞）。进一步的分析表明，Mo/MΦ 存在两个亚群，包括 M1 和 M2 型，B 细胞存在四个亚群，即成熟 B 细胞、未成熟 B 细胞、前 B 细胞和早期前 B 细胞。我们的研究将为理解硬骨鱼类免疫细胞群体的生物学特性、发育和功能提供新的线索。此外，我们的方法为目前没有参考基因组的硬骨鱼类 scRNA-seq 数据分析提供了可靠的替代方法。
