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

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 () and Yi-Zhi Liu ()
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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

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38324-9

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