Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.
Center for Cancer Genomics, Robert H. Lurie Cancer Center, Northwestern University, Chicago, IL, 60611, USA.
Nat Commun. 2023 Jul 11;14(1):4124. doi: 10.1038/s41467-023-39813-7.
Single-cell nanopore sequencing of full-length mRNAs transforms single-cell multi-omics studies. However, challenges include high sequencing errors and dependence on short-reads and/or barcode whitelists. To address these, we develop scNanoGPS to calculate same-cell genotypes (mutations) and phenotypes (gene/isoform expressions) without short-read nor whitelist guidance. We apply scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell-lines. Standalone, scNanoGPS deconvolutes error-prone long-reads into single-cells and single-molecules, and simultaneously accesses both phenotypes and genotypes of individual cells. Our analyses reveal that tumor and stroma/immune cells express distinct combination of isoforms (DCIs). In a kidney tumor, we identify 924 DCI genes involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Transcriptome-wide mutation analyses identify many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting the critical roles of different mutant populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing technologies.
全长 mRNA 的单分子纳米孔测序技术变革了单细胞多组学研究。然而，该技术仍面临测序错误率高、依赖短读长和/或条码白名单等挑战。为解决这些问题，我们开发了 scNanoGPS 技术，在无需短读长和白名单指导的情况下，计算同一细胞的基因型（突变）和表型（基因/异构体表达）。我们将 scNanoGPS 应用于 4 个肿瘤和 2 个细胞系的 23587 个长读长转录组。scNanoGPS 可以将易错的长读长单独解析为单细胞和单分子，同时获取单个细胞的表型和基因型。我们的分析表明，肿瘤和基质/免疫细胞表达不同的异构体组合（DCIs）。在一个肾肿瘤中，我们鉴定出 924 个与细胞类型特异性功能相关的 DCI 基因，如肿瘤细胞中的 PDE10A 和淋巴细胞中的 CCL3。全转录组突变分析鉴定出许多细胞类型特异性的突变，包括肿瘤细胞中的 VEGFA 突变和免疫细胞中的 HLA-A 突变，突出了不同突变群体在肿瘤中的关键作用。总之，scNanoGPS 促进了单细胞长读长测序技术的应用。
