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.

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

“…However, this problem can be overcome via the combined usage of dRNA-seq and 5′-end sensitive PCR-free direct cDNA sequencing methods (dcDNA) 22 , 27 – 29 . Furthermore, direct cDNA-seq can be used to accurately quantify gene expression, as it is not affected by biases introduced in the RT-PCR of traditional PCR-cDNA-sequencing 30 .…”

“…There are several bioinformatic tools that can be used to achieve this, including: TALON 50 ; LIQA 51 ; LoRTIA ( https://github.com/zsolt-balazs/LoRTIA ); EPI2ME’s transcriptomes workflow ( https://github.com/epi2me-labs/wf-transcriptomes ) or SQUANTI3 ( https://github.com/ConesaLab/SQANTI3 52 ). Transcript annotation can be carried out from both types of sequencing data (dcDNA and dRNA), however as dRNA-seq yields less artificial or false products, it is suggested to use these reads for validating the dcDNA-seq derived transcripts 30 . Although it is possible that some rare transcripts that are expressed in a subset of the time-points exclusively (e.g., some immediate early isoforms) could not be captured in the dRNA sequencing library.…”

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

“…This problem can be circumvented with the combined usage of 5'-end sensitive PCR-free direct cDNA sequencing methods (dcDNA) 23,[28][29][30] . Moreover, direct cDNA-seq can be used to accurately quantify gene expression, as it is not affected by biases introduced in the RT-PCR of traditional PCR-cDNAsequencing 31 .…”

“…There are several bioinformatic tools that can be used to achieve this, including: TALON 45 ; LIQA 46 ; LoRTIA (https://github.com/zsolt-balazs/LoRTIA); EPI2ME's transcriptomes workflow (https://github.com/epi2me-labs/wf-transcriptomes) or SQUANTI3 (https://github.com/ConesaLab/SQANTI3, 47 ). Transcript annotation can be carried out from both types of sequencing data (dcDNA and dRNA), however as dRNA-seq yields less artificial or false products, it is suggested to use these reads for validating the dcDNA-seq derived transcripts 31 . Although it is possible that some rare transcripts that are expressed in a subset of the time-points exclusively (e.g., some immediate early isoforms) could not be captured in the dRNA sequencing library.…”

Novel Herpesvirus Transcripts with Putative Regulatory Roles in DNA Replication and Global Transcription

“…In HSV-1, we also detected an ICP4 TIs with long 5′ UTR, which overlaps the us1 gene. Additionally, a 3′ UTR isoform of icp4 has been described to form a parallel TO with the downstream icp0 gene in BoHV-1, however, the icp4 ORF is spliced out from this transcript resulting a chimeric transcript containing the full-length icp0 gene and part of the 5′ UTR of ICP4 transcript 79 . Additionally, ICP4 TIs were detected to form similar chimeric transcripts and also bicistronic transcripts with the BoHV-1 CIRC RNA mapping to the adjacent genomic locus in the circular or concatemeric viral genome.…”