Virome Assembly and Annotation: A Surprise in the Namib Desert

Abstract

Sequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ≥50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies.

Namib Desert Soil Microbial Community Diversity, Assembly, and Function Along a Natural Xeric Gradient

“…are common in Namib Desert soils (Makhalanyane et al, 2015) and Yersina phages have already been detected in desert soils (Prestel et al, 2013), the detection of the marine Cellulophaga phage phi19:1 (Sepulveda et al, 2016) throughout the transect was unexpected. Marine-phage sequences have recently been detected in a ~100km inland Namib metaviromic study (Hesse et al, 2017), and our result, therefore, tends to confirm their hypothesis that marine fog and wind play a role in the dispersal of (marine) phages in Namib Desert soils.…”

Choice of assembly software has a critical impact on virome characterisation

“…Normalised genome abundance of 572 members of a published simulated community, (Fig. 1a) [20] the proportion of genome recovered and the degree of fragmentation were assessed by aligning the resulting contigs from each assembler to the reference genomes (Fig. 1b).…”

“…Although these simulated datasets are undergoing constant improvements [13, 48], they have focused primarily on bacterial metagenomes and remain limited in their ability to accurately replicate the challenges of true metagenomes. While some virome-specific assembly benchmarks have been performed, many have been limited to a small number of assemblers, 454 pyrosequencing data or subsections of virome studies which have exclusively used simulated data [3, 14, 20, 44, 51, 53].…”

Novel ssDNA Viruses Detected in the Virome of Bleached, Habitat-Forming Kelp Ecklonia radiata

“…SPAdes was chosen as it performs better than other assemblers on virome data (Hesse et al, 2017;Papudeshi et al, 2017;Vollmers et al, 2017). Reads were mapped to the contigs using Bowtie 2.2.5 (Langmead and Salzberg, 2012) and visualized using Tablet (Milne et al, 2009) to assess the evenness of read coverage and to identify possible chimeric assemblies, which were removed from further analysis.…”