HIT-scISOseq: High-throughput and High-accuracy Single-cell Full-length Isoform Sequencing

Abstract Although long-read single-cell isoform sequencing (scISO-Seq) can reveal transcriptomic dynamics in individual cells invisible to NGS-based single-cell RNA analysis, scISO-Seq has been limited by low throughput, high error rates, and long running time.Here, we introduce HIT-scISOseq, the first method that concatenates multiple full-length cDNAs for PacBio circular consensus sequencing (CCS) sequencing to achievehigh-throughput, and high-accuracy single-cell isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy full-length isoforms in a single PacBio Sequel II SMRT Cell 8M. We have developed scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell full-length isoforms with >99.99% accuracy and specificity. We have applied HIT-scISOseq to characterize the transcriptome of thousands of corneal limbus cells, and reveal cell-type-specific isoform expression changes that are previously not identified by NGS-based scRNAseq. HIT-scISOseq is a high-throughput, high-accuracy, and technically accessible method that can be used by most laboratories to accelerate the burgeoning field of long-read single-cell transcriptomics.