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

Cheng Kai Shiau; Lina Lu; Rachel Kieser; Kazutaka Fukumura; Timothy Pan; Hsiao Yun Lin; Jie Yang; Eric L. Tong; Ga Hyun Lee; Yuanqing Yan; Jason T. Huse; Ruli Gao

doi:10.1038/s41467-023-39813-7

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

Cheng Kai Shiau, Lina Lu, Rachel Kieser, Kazutaka Fukumura, Timothy Pan, Hsiao Yun Lin, Jie Yang, Eric L. Tong, Ga Hyun Lee, Yuanqing Yan, Jason T. Huse, Ruli Gao^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

25 Scopus citations

Abstract

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.

Original language	English (US)
Article number	4124
Journal	Nature communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-39813-7
State	Published - Dec 2023

Funding

We thank research funding supports to R.G. from Northwestern University and National Heart, Lung, and Blood Institute (NIH 1R01HL160552-01). The nanopore sequencing was carried out by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant (1S10OD010786-01). The next-generation sequencing was carried out by the ATGC core at the University of Texas MD Anderson Cancer Center, supported by the Core grant (CA016672) and NIH Shared Instrument Grant (1S10OD024977-01), and by the Genomic and RNA Profiling Core at Baylor College of Medicine with funding support from an NIH S10 grant (1S10OD023469).

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

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10.1038/s41467-023-39813-7

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

abstract = "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.",

author = "Shiau, {Cheng Kai} and Lina Lu and Rachel Kieser and Kazutaka Fukumura and Timothy Pan and Lin, {Hsiao Yun} and Jie Yang and Tong, {Eric L.} and Lee, {Ga Hyun} and Yuanqing Yan and Huse, {Jason T.} and Ruli Gao",

note = "Funding Information: We thank research funding supports to R.G. from Northwestern University and National Heart, Lung, and Blood Institute (NIH 1R01HL160552-01). The nanopore sequencing was carried out by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant (1S10OD010786-01). The next-generation sequencing was carried out by the ATGC core at the University of Texas MD Anderson Cancer Center, supported by the Core grant (CA016672) and NIH Shared Instrument Grant (1S10OD024977-01), and by the Genomic and RNA Profiling Core at Baylor College of Medicine with funding support from an NIH S10 grant (1S10OD023469). Publisher Copyright: {\textcopyright} 2023, The Author(s).",

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doi = "10.1038/s41467-023-39813-7",

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AU - Pan, Timothy

AU - Lin, Hsiao Yun

AU - Yang, Jie

AU - Tong, Eric L.

AU - Lee, Ga Hyun

AU - Yan, Yuanqing

AU - Huse, Jason T.

AU - Gao, Ruli

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N2 - 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.

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

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