Dataset Information

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

ABSTRACT: In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate-early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.

SUBMITTER: Tombacz D

PROVIDER: S-EPMC9229804 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

Tombácz Dóra D Kakuk Balázs B Torma Gábor G Csabai Zsolt Z Gulyás Gábor G Tamás Vivien V Zádori Zoltán Z Jefferson Victoria A VA Meyer Florencia F Boldogkői Zsolt Z

Viruses 20220613 6

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an ...[more]

PMID: 35746760

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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.

Dataset Information

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

Publications

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

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