Enhanced Viral Metagenomics with Lazypipe 2

Abstract

Viruses are the main agents causing emerging and re-emerging infectious diseases. It is therefore important to screen for and detect them and uncover the evolutionary processes that support their ability to jump species boundaries and establish themselves in new hosts. Metagenomic next-generation sequencing (mNGS) is a high-throughput, impartial technology that has enabled virologists to detect either known or novel, divergent viruses from clinical, animal, wildlife and environmental samples, with little a priori assumptions. mNGS is heavily dependent on bioinformatic analysis, with an emerging demand for integrated bioinformatic workflows. Here, we present Lazypipe2, an updated mNGS pipeline with, as compared to Lazypipe1, significant improvements in code stability and transparency, with added functionality and support for new software components. We also present extensive benchmarking results, including evaluation of a novel canine simulated metagenome, precision and recall of virus detection at varying sequencing depth, and a low to extremely low proportion of viral genetic material. Additionally, we report accuracy of virus detection with two strategies: homology searches using nucleotide or amino acid sequences. We show that Lazypipe2 with nucleotide-based annotation approaches near perfect detection for eukaryotic viruses and, in terms of accuracy, outperforms the compared pipelines. We also discuss the importance of homology searches with amino acid sequences for the detection of highly divergent novel viruses.

Virus Pop—Expanding Viral Databases by Protein Sequence Simulation

“…The purpose was to illustrate how the local alignment performance varies with simulated distances and how different tools yield different performances. We chose BLASTp and DIAMOND as they are widely used for local sequence alignment of protein sequences and are often integrated within taxonomic assignation pipelines (e.g., [ 1 , 2 , 3 ]). Depending on the software used within each taxonomic assignation pipeline, their parameters and cut-off values, their performances will be affected in a different way.…”

“…In virology, metagenomics tools performing taxonomic assignation from high throughput sequencing datasets are currently key for detecting unknown viruses and improving our knowledge of the virosphere (e.g., [ 1 , 2 , 3 ]). They are generally evaluated with datasets derived from biological samples or in silico spiked samples, which all contain known viral sequences present in public databases.…”

Equine dermatitis outbreak associated with parapoxvirus

“…We used DNA extracted from swab samples previously [12] and most of the samples had already been tested with pan-PPV-PCR described by Inoshima et al [14]. Six samples from five stables were subjected to next generation sequencing (NGS) with previously described protocol [12] and assembled with LazyPipe2 [18, 19]. Genes were annotated with Open Reading Frame Finder (NCBI) with minimum ORF length of 150 nucleotides.…”

Exploring Cereal Metagenomics: Unravelling Microbial Communities for Improved Food Security

“… Workflow for high throughput metagenomics (amplicon and shotgun) data analysis [ 85 , 86 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 , 187 , 188 , 189 , 190 ]. …”