DEN-IM: Dengue Virus identification from shotgun and targeted metagenomics

Abstract

Dengue virus (DENV) represents a public health and economic burden in affected countries. The availability of genomic data is key to understand viral evolution and dynamics, supporting improved control strategies. Currently, the use of High Throughput Sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR amplified viral sequences (targeted metagenomics), is the most informative approach to monitor the viral dissemination and genetic diversity.Despite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper reproducibility and comparison of results.Here we present DEN-IM, a one-stop, user-friendly, containerised and reproducible workflow for the analysis of DENV sequencing data, both from shotgun and targeted metagenomics approaches. It is able to infer DENV coding sequence (CDS), identify serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement of extensive bioinformatics expertise.Using DEN-IM, we successfully analysed two DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples of varying serotype and genotype, including a spiked sample containing the existing four serotypes. The second dataset consisted of 106 targeted metagenomics samples of DENV 3 genotype III where DEN-IM allowed detection of the intra-genotype diversity.The DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM. The Dengue virus (DENV), a single-stranded positive-sense RNA virus belonging to the Flavivirus 2 genus, is one of the most prevalent arboviruses and is mainly concentrated in tropical and subtropical 3 regions. Infection with DENV results in symptoms ranging from mild fever to haemorrhagic fever and 4 shock syndrome [1]. Transmission to humans occurs through the bite of Aedes mosquitoes namely 5 Aedes aegypti and Aedes albopictus [2] In 2010, it was predicted that the burden of dengue disease 6 reached 390 million cases per year worldwide [3]. The high morbidity and mortality of dengue makes 7 it the arbovirus with the highest clinical significance [4]. 8 The viral genome of ∼11,000 nucleotides, consists of a Coding Sequence (CDS) of approximately 910.2 Kb that is translated into a single polyprotein encoding three structural proteins (capsid -C, 10 premembrane -prM, envelope -E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, 11 NS4B and NS5. Additionally, the genome contains two Non-Coding Regions (NCRs) at their 5' and 12 3' ends [5]. 13DENV can be classified into four serotypes (1, 2, 3 and 4), differing from each other by 25% to 14 40% at the amino acid level. They are further classified into genotypes that vary by up to ∼...