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

Abstract

The recent human Monkeypox outbreak underlined the importance of studying 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. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

Fig. 1

General overview of the study. Briefly, MPXV was isolated from a skin lesion and then was used to infect CV-1 cells. After the designated infection times, total RNA was isolated and sequenced using direct cDNA sequencing protocol on ONT’s MinION platform. The experiment was carried out in triplicates. A mixed time-point sample was also prepared and used for direct RNA sequencing. The reads were basecalled and then mapped to the viral and host genomes. From the alignments viral coverages were calculated and visualized. The figure was created with Biorender (BioRender.com).

Fig. 2

Sequencing read counts and viral read ratios in the dcDNA samples. In the left panel purple dots represent the number of viral reads, while green dots represent the number of host reads in each biological replicate. In the right panel, the dots represent the ratio of viral reads to the total read count per sample. The colored lines in both panels represent the result of a smoothing function, while the grey lines represent 95% confidence intervals. A clear decrease in the host reads and an increase in the viral read ratio shows the progress of the viral infection.

Fig. 3

Violin plot iluustration of the read length distributions in the cDNA and the dRNA sequencing libraries. The hinges of the added boxes correspond to the first and third quartiles of the data, and the bold line indicating the median values.

Fig. 4

Coverage of the viral genome in the dRNA sequencing library. The mean coverage on the monkeypox genome was calculated in a 100-nt window. The links in the center of the circle represent transcripts, as in the connections between the 5′- and 3′-ends of the reads. These potential ‘transcripts’ were filtered to read count threshold of 10. The transparency of the links is correlated with the abundance of the ‘transcripts’.

Fig. 5

Log10 transformed coverage of the viral genome in the dcDNA sequencing library. (a) Coverage in 1-, 2- and 4-hours post-infection; (b) coverage in 6-, 12- and 24-hours post-infection.

Fig. 6

Quality of total RNA samples. The quality of the RNAs were assessed by using a TapeStation 4150 System and RNA ScreenTape (both from Agilent Technologies). TapeStation gel image shows that intact, high-quality RNAs (RIN > 9) were isolated from the cells and used for Nanopore sequencing. The image shows the following samples: EL1(L): marker; A1: 1 h (replicate); B1: 1 h (replicate C); C1: 2 h (replicate A); D1: 2 h (replicate B); E1: 4 h (replicate A); F1: 4 h (replicate B); G1: 6 h (replicate A); H1: 6 h (replicate B); A2: 12 h (replicate A); B2: 12 h (replicate C); C2: 24 h (replicate A); D2: 24 h (replicate B); E2: 2 h (used for dRNA-seq); F2: 6 h (used for dRNA-seq); G2: 12 h (for dRNA-seq); H2: 24 h (for dRNA-seq).