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

• Published in: Nature communications Vol. 14; no. 1; pp. 2631 - 13
• Main Authors: Shi, Zhuo-Xing, Chen, Zhi-Chao, Zhong, Jia-Yong, Hu, Kun-Hua, Zheng, Ying-Feng, Chen, Ying, Xie, Shang-Qian, Bo, Xiao-Chen, Luo, Feng, Tang, Chong, Xiao, Chuan-Le, Liu, Yi-Zhi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.05.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 38/91; 49/39; 631/1647/2163; 631/1647/2217/2218; 631/1647/514/1949; 631/1647/514/2254; 631/337/2019; Accuracy; Alternative splicing; Consensus; Cornea; Gene sequencing; High-Throughput Nucleotide Sequencing - methods; Humanities and Social Sciences; multidisciplinary; Protein Isoforms - genetics; Ribonucleic acid; RNA; RNA Isoforms - genetics; Science; Science (multidisciplinary); Sequence Analysis, DNA - methods; Sequence Analysis, RNA; Splicing; Transcriptomes; Transcriptomics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-38324-9

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. Long-read single-cell RNA isoform sequencing can elucidate the intricate landscape of alternative RNA splicing in individual cells, but it suffers from a low read throughput. Here, the authors develop circular consensus sequencing methods to allow high-throughput and high-accuracy single-cell RNA isoform sequencing.