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

作者：Zhuoxing Shi, Zhichao Chen, Jiayong Zhong, Kun-Hua Hu, Yingfeng Zheng, Ying Chen, Shuang Xie, Xiaochen Bo, Feng Luo, Chong Tang, Chuan‐Le Xiao, Yizhi Liu · 发表于：Nature Communications · 年份：2023 · DOI：10.1038/s41467-023-38324-9 · 被引用次数：67 · 研究领域：Single-cell and spatial transcriptomics、Cancer-related molecular mechanisms research、RNA modifications and cancer

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.