Denes, B. | bioRxiv Science

bioRxiv2022

In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing

The recent Monkeypox outbreak showed the importance of studying the basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. Our direct cDNA and native RNA sequencing data enable the in-depth characterization of the transcriptomic architecture and dynamics of the gene expressions of monkeypox virus; and also the deeper understanding of the changes it causes in the host cells on a transcriptome level.

microbiology↗

bioRxiv2021

High-Spatiotemporal-Resolution Nanopore Sequencing of SARS-CoV-2 and Host Cell RNAs

Recent studies have disclosed the genome, transcriptome and epigenetic compositions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the effect of viral infection on gene expression of the host cells. It has been demonstrated that, besides the major canonical transcripts, the viral genome also codes for non-canonical RNA molecules. While the structural characterizations have revealed a detailed transcriptomic architecture of the virus, the kinetic studies provided poor and often misleading results on the dynamics of both the viral and host transcripts due to the low temporal resolution of the infection event and the low virus/cell ratio (MOI=0.1) applied for the infection. In this study, we used direct cDNA and direct RNA nanopore sequencings for the generation of high-coverage, high-temporal-resolution transcriptomic datasets on SARS-CoV-2 and on primate host cells infected with a high virus titer (MOI=5). Sixteen sampling time points ranging from 1 to 96h with a varying time resolution and three biological replicates were used in the experiment for both the infected and the non-infected cells.

genomics↗

bioRxiv2020

Long-read Assays Shed New Light on the Transcriptome Complexity of a Viral Pathogen and on Virus-Host Interaction

Characterization of global transcriptomes using conventional short-read sequencing is challenging because of the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps, etc. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the dynamic vaccinia virus (VACV) transcriptome and assess the effect of viral infection on host gene expression. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighboring genes. We distinguished five classes of host genes according to their temporal responses to viral infection. This study provides novel insights into the transcriptomic profile of a viral pathogen and the effect of the virus on host gene expression. Author SummaryViral transcriptomes that are determined using conventional (first- and second-generation) sequencing techniques are incomplete because these platforms are inefficient or fail to distinguish between types of transcripts and transcript isoforms. In particular, conventional sequencing techniques fail to distinguish between parallel overlapping transcripts, including alternative polycistronic transcripts, transcriptional start site (TSS) and transcriptional end site (TES) isoforms, and splice variants and RNA molecules that are produced by transcriptional read-throughs. Long-read sequencing (LRS) can provide complete sets of RNA molecules, and can therefore be used to assemble complete transcriptome atlases of organisms. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome contains large numbers of TSSs and TESs for individual viral genes and has a high degree of polycistronism, together leading to enormous complexity. In this study, we applied single molecule real-time and nanopore-based cDNA and direct-RNA sequencing methods to investigate transcripts of VACV and the host organism.

genomics↗