INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillance

Abstract

Background: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification.

Results: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis.

Conclusions: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).

Fig. 1

Architecture of the INSaFLU-TELEVIR platform, summarizing the implemented analytical modules for viral metagenomic detection (TELEVIR) and routine genomic surveillance (INSaFLU and NextStrain)

Fig. 2

Simplified illustration of the main steps of the modular INSaFLU-TELEVIR bioinformatics pipeline for metagenomics virus detection (TELEVIR). Documentation for each step is provided at the website (https://insaflu.insa.pt)

Fig. 3

Simplified illustration of the benchmark of the virus identification pipeline (TELEVIR) module components, which is described in detail in Additional file 1. A Tree representation of module combinations. From left to right, sections represent pipeline steps (exemplified for Illumina) as followed at runtime: (1) Quality Control, (2) Viral Enrichment, (3) Assembly, (4) Contig Classification, (5) Read Classification. Nodes represent software, parameters, or databases compared. Color gradient corresponds to the product of four assessment statistics: mapped reads proportion, horizontal coverage, true positive rate, and completeness (proportion of hits with both read and contig evidence). Statistics were standardized by their respective maxima. B Heatmap representation of software benchmarked for Illumina samples, parameters not discriminated, color code at the bottom. C Table of individual statistics for each node, standardized across samples as in A. For panels A and C, darker colors = lower values, lighter colors = higher values

Fig. 4

Snapshot of dashboard reports of the INSaFLU-TELEVIR bioinformatics module for metagenomics virus detection. Interactive examples are available at the https://insaflu.insa.pt/ [22] through an open “demo” account

Fig. 5

Rapid, robust, and cost-efficient diagnostics using findONTime in combination with MinION sequencing. A simulated scenario of hypothesis-free ONT sequencing using data from a MPXV-positive sample, prepared without prior viral enrichment/host depletion. The plot shows the number of reads mapping to a MPXV reference genome and the percentage of horizontal coverage at increasing time points, during the sequencing run. Reference genome identified with over 50% coverage after 20 min. Contigs mapped at the 40-min mark. Strong evidence (mapped contigs; > 90% reference genome covered by at least one read) is achieved in under 2 h (1 h 20 min)