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High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing

Author

Listed:
  • Zhuo-Xing Shi

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science)

  • Zhi-Chao Chen

    (University of Chinese Academy of Sciences)

  • Jia-Yong Zhong

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science)

  • Kun-Hua Hu

    (the Third Affiliated Hospital of Sun Yat-sen University)

  • Ying-Feng Zheng

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science)

  • Ying Chen

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science)

  • Shang-Qian Xie

    (Hainan University)

  • Xiao-Chen Bo

    (Beijing Institute of Radiation Medicine)

  • Feng Luo

    (Clemson University)

  • Chong Tang

    (BGI Genomics, BGI Shenzhen)

  • Chuan-Le Xiao

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science)

  • Yi-Zhi Liu

    (Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science
    Chinese Academy of Medical Sciences)

Abstract

Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.

Suggested Citation

  • Zhuo-Xing Shi & Zhi-Chao Chen & Jia-Yong Zhong & Kun-Hua Hu & Ying-Feng Zheng & Ying Chen & Shang-Qian Xie & Xiao-Chen Bo & Feng Luo & Chong Tang & Chuan-Le Xiao & Yi-Zhi Liu, 2023. "High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38324-9
    DOI: 10.1038/s41467-023-38324-9
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    References listed on IDEAS

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    1. Ashley Byrne & Anna E. Beaudin & Hugh E. Olsen & Miten Jain & Charles Cole & Theron Palmer & Rebecca M. DuBois & E. Camilla Forsberg & Mark Akeson & Christopher Vollmers, 2017. "Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells," Nature Communications, Nature, vol. 8(1), pages 1-11, December.
    2. Kevin Lebrigand & Virginie Magnone & Pascal Barbry & Rainer Waldmann, 2020. "High throughput error corrected Nanopore single cell transcriptome sequencing," Nature Communications, Nature, vol. 11(1), pages 1-8, December.
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    Cited by:

    1. Arthur Dondi & Ulrike Lischetti & Francis Jacob & Franziska Singer & Nico Borgsmüller & Ricardo Coelho & Viola Heinzelmann-Schwarz & Christian Beisel & Niko Beerenwinkel, 2023. "Detection of isoforms and genomic alterations by high-throughput full-length single-cell RNA sequencing in ovarian cancer," Nature Communications, Nature, vol. 14(1), pages 1-19, December.

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