High-throughput and high-sensitivity full-length single-cell RNA-seq analysis on third-generation sequencing platform
Yuhan Liao, Zhenyu Liu, Yu Zhang, Ping Lü, Lu Wen, Fuchou Tang
Abstract
The advancement of single-cell RNA-seq technologies based on third-generation sequencing (TGS) platforms has accelerated biological researches. Several TGS platform-based single-cell RNA-seq methods have been developed since 2016 1 . Limited by low accuracy and sensitivity, they either combine NGS platform-based methods to reduce the error rates 2 , or sacrifice the throughput to improve detection rates 3 . To overcome these drawbacks, here we developed SCAN-seq2, a high-throughput, high-sensitivity full-length single-cell RNA-seq method based on TGS platform. We performed SCAN-seq2 to a total of 5472 cells from nine cell lines. Detailed flowchart of the data processing pipeline and overall statistics of reads after filtering, demultiplexing and deduplication were shown (Supplementary Figs. S1 and S2 ). Through reference-guided transcriptome assembly, we identified thousands of novel full-length RNA isoforms. Transcripts of pseudogenes could be distinguished from transcripts of corresponding parent genes and hundreds of them showed cell-type-specific expression patterns. Moreover, we showed that V(D)J rearrangement events could be accurately determined for the highly polymorphic T cell receptor (TCR) and B cell receptor (BCR) genes (immunoglobins). Finally, we demonstrated the conserved apoptosis response of HepG2 and Hela cells after treated by spliceosome inhibitor Isoginkgetin (IGG). SCAN-seq2 proves to be a new promising tool for single cell full-length transcriptome research.