High throughput single cell long-read sequencing analyses of same-cell genotypes and phenotypes in human tumors

• Published in: Nature communications Vol. 14; no. 1; pp. 4124 - 12
• Main Authors: Shiau, Cheng-Kai, Lu, Lina, Kieser, Rachel, Fukumura, Kazutaka, Pan, Timothy, Lin, Hsiao-Yun, Yang, Jie, Tong, Eric L., Lee, GaHyun, Yan, Yuanqing, Huse, Jason T., Gao, Ruli
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.07.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 38/23; 38/39; 45/90; 45/91; 631/114/2785/2302; 631/114/794; 631/208/1792; 631/208/514/1949; 631/61/212/2019; Bar codes; Carcinoma, Intraductal, Noninfiltrating; Genotype; Genotypes; High-Throughput Nucleotide Sequencing; Humanities and Social Sciences; Humans; Immune system; Isoforms; Kidney Neoplasms; Lymphocytes; multidisciplinary; Mutation; Phenotype; Phenotypes; Phosphoric Diester Hydrolases; Science; Science (multidisciplinary); Stroma; Transcriptomes; Tumor cells; Tumors
• ISSN: 2041-1723; 2041-1723
• 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. There is a need for methods that allow the analysis of single-cell long-read sequencing data without depending on known barcode lists or short-read sequencing. Here, the authors develop scNanoGPS, a tool that can independently deconvolute long reads into single cells and single molecules, and apply it on tumour and cell line data.