Journal ArticleHigh-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencingShi, 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-ZhiAbstract 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.2023-12-0110416832Nature Communications1412041-1723https://doi.org/10.1038/s41467-023-38324-91759856; 2025541National Science Foundation