Pox virus genome analysis from tiled-amplicon sequencing data

Sammanfattning

Pox viruses (Poxviridae) are a large family of viruses, and members of it have various vertebrate and arthropod species as their natural hosts. The most widely known species in the family are the now extinct variola virus from the genus orthopoxvirus as the cause of smallpox, and vaccinia virus, a related, likely horsepox virus, which served as the source for the smallpox vaccine that allowed eradication of that disease.

However, various other pox viruses infect mammalian hosts and cause often severe diseases, and some of them have the ability to jump to humans. Mpox, caused by monkeypox virus, is the most recent and prominent example demonstrating the zoonotic potential of orthopoxviruses. The sister genus capripoxvirus is a major concern in livestock farming with all its three species - sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) causing severe disease in sheep and goats (SPPV, GTPV) and in cattle (LSDV).

All pox viruses are characterized by a large double-stranded DNA genome (genome sizes of currently sequenced species range from 135 to 360kb) that gets replicated exclusively in the cytoplasm of host cells.

The ends of pox virus genomes consist of inverted terminal repeats (ITRs), i.e. regions of exact reverse-complementarity at the 5’- and 3’-end of the genome. At the very tips of the genome, these regions contain tandem repeats and terminal bases that are cross-linked to the opposite genome strand as first described for vaccinia virus and reviewed in Wittek 1982.

There is considerable length variation in the ITR regions of different species. For example, Capripox virus genomes have ITR lengths of 2.2 - 2.4 kb, monkeypox virus has 6.4 kb long ITRs, and variola virus had the shortest known ITRs with a length of just 700 bases (reviewed in Haller et al. 2014).

Independent of their size, ITRs pose a problem for the analysis of high-throughput sequencing data of pox viruses. Their repetitive nature can confuse de-novo assembly algorithms, but is, to some extent, also a challenge during reference-based mapping, particularly at the boundary between ITRs and the central region of the genome and with samples less related to the reference.

Recently, a tiled-amplicon approach with separate sequencing of half-genomes has been developed for capripoxviruses (Mathijs et al. 2022) that makes it possible to assign ITR-derived reads unambiguously to the 5’- or 3’-end of a pox virus genome when combined with a suitable bioinformatics pipeline.

This tutorial demonstrates how such data can be analyzed correctly with Galaxy.

Publicerad i

Utgivare: Galaxy Training

SLU författare

• Klingström, Tomas

  • Institutionen för husdjurens biovetenskaper, Sveriges lantbruksuniversitet
    

UKÄ forskningsämne

Bioinformatik (beräkningsbiologi)
Mikrobiologi inom det medicinska området

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/139593