Shallow nanopore RNA sequencing enables transcriptome profiling for precision cancer medicine

Abstract

Transcriptome profiling is a mainstay of translational cancer research and is increasingly finding its way into precision oncology. While bulk RNA sequencing (RNA-seq) is widely available, high costs and long data return time are limiting factors for clinical applications. We investigated a portable nanopore long-read sequencing device (MinION, Oxford Nanopore Technologies) for transcriptome profiling of tumors. In particular, we investigated the impact of lower coverage than that of larger sequencing devices by comparing shallow nanopore RNA-seq data with short-read RNA-seq data generated using reversible dye terminator technology (Illumina) for ten samples representing four cancer types. Coupled with ShaNTi (Shallow Nanopore Sequencing for Transcriptomics), a newly developed data processing pipeline, a turnaround time of five days was achieved. The correlation of normalized gene-level counts between nanopore and Illumina RNA-seq was high for MinION but not for very low-throughput Flongle flow cells (r = 0.89 and r = 0.24, respectively). A cost-saving approach based on multiplexing of four samples per MinION flow cell maintained a high correlation with Illumina data (r = 0.56 - 0.86). In addition, we compared the utility of nanopore and Illumina RNA-seq data for analysis tools commonly applied in translational oncology: (i) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (ii) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (iii) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (iv) Shallow nanopore RNA-seq was successfully applied to validate known gene fusions by breakpoint analysis. These findings suggest that shallow nanopore RNA-seq enables rapid, cost-effective, and biologically meaningful transcriptome profiling of tumors and warrants further exploration in precision cancer medicine studies.