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Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing.

ABSTRACT: Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.

SUBMITTER: Maroti Z

PROVIDER: S-EPMC8270970 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 investment 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: (1) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (2) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (3) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (4) Shallow nanopore RNA-seq was successfully applied to detect fusion genes using the JAFFAL pipeline. These findings suggest that shallow nanopore RNA-seq enables rapid and biologically meaningful transcriptome profiling of tumors, and warrants further exploration in precision cancer medicine studies.

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Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing.

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