In an age where pathogens continue to evolve and cross boundaries, understanding what makes them virulent—that is, capable of causing disease—has become a critical focus in modern microbiology and genomics. Virulence prediction bridges computational biology, genomics, and machine learning to forecast the pathogenic potential of microbes before they strike.

What Is Virulence?

Virulence refers to the degree of damage a pathogen can inflict on its host. It is determined by a combination of genetic factors—called virulence factors (VFs)—that allow the organism to attach, invade, evade, and harm the host. These include genes coding for toxins, secretion systems, adhesins, and enzymes that disrupt host defenses.

Understanding virulence factors not only helps in deciphering the mechanisms of infection but also provides early warning signs for emerging threats.

Why Predict Virulence?

Traditional virulence studies relied heavily on experimental infection models, which, although accurate, are time-consuming, expensive, and ethically constrained.
Today, the availability of whole-genome sequences and large-scale pathogen databases has paved the way for in silico virulence prediction—a computational approach that can screen thousands of genomes within hours.

This approach enables researchers to:

• Rapidly identify potential high-risk strains.

• Prioritize pathogens for containment, surveillance, or further study.

• Guide vaccine development and drug target discovery.

• Support One Health frameworks, linking animal, human, and environmental health data.

How Is Virulence Predicted?

Virulence prediction combines bioinformatics pipelines with machine learning and comparative genomics. The process generally involves:

1. Genome Annotation: Identifying genes and coding sequences in microbial genomes.

2. Feature Extraction: Comparing sequences with curated databases like VFDB (Virulence Factor Database), PATRIC, or Victors.

3. Pattern Recognition: Using algorithms (e.g., Random Forest, SVM, or deep learning models) to classify genes or strains as virulent or non-virulent based on sequence patterns, motifs, and protein domains.

4. Scoring and Visualization: Assigning a virulence score or confidence level and visualizing it through heatmaps or genome maps.

Tools and Resources for Virulence Prediction

A number of tools and databases make virulence prediction accessible to the scientific community:

• VFanalyzer – For identifying virulence genes based on VFDB.

• PathoFact – Predicts virulence, antimicrobial resistance (AMR), and toxin genes from metagenomic data.

• Pangenome-based models – Identify virulence-associated gene clusters across strains.

• Machine learning models – Use features like GC content, codon usage bias, or protein domains to predict pathogenicity.

Emerging tools now integrate multi-omic data—including transcriptomics, proteomics, and metabolomics—to understand virulence in a systems biology framework.

Applications in the Real World

Virulence prediction has major implications across public health and research sectors:

• Epidemic preparedness: Early identification of virulent strains in outbreak samples.

• AMR surveillance: Linking virulence profiles with antibiotic resistance determinants.

• Environmental monitoring: Predicting pathogenic potential of soil or waterborne microbes.

• Clinical diagnostics: Supporting personalized treatment through pathogen profiling.

For instance, integrating virulence prediction pipelines into national surveillance networks could enable faster risk assessment and response to infectious outbreaks.

The Road Ahead

As machine learning and genomics advance, virulence prediction will evolve from simple gene-based detection to dynamic, context-aware models that account for host–pathogen interactions, environmental signals, and evolutionary adaptation.

Future tools may predict not just if a strain is virulent, but under what conditions it expresses that virulence—bridging the gap between genotype and phenotype.

In Summary

Virulence prediction is redefining how we understand and anticipate infectious diseases. By coupling genomic insights with computational intelligence, researchers can identify potential threats earlier, design smarter interventions, and ultimately, strengthen our preparedness against emerging pathogens.

kallisto

Following are the list of standalone applications for network analysis:

Arena 3D

3D visualization of multi-layer networks

http://www.arena3d.org

Biana

Data integration and network management

http://sbi.imim.es/web/BIANA.php

BioLayout Express 3D 

2D/3D network visualization

http://www.biolayout.org/

BiologicalNetworks 

Efficient integrated multi-level analysis of microarray, sequence, regulatory and other data

http://www.biologicalnetworks.org

BioMiner

Modeling, analyzing and visualizing biochemical pathways and networks

http://www.zbi.uni-saarland.de/chair/projects/BioMiner

Cell Illustrator 

Petri nets for modeling and simulating biological networks

http://www.cellillustrator.com

COPASI

Analysis of biochemical networks and their dynamics

http://www.copasi.org/

Cytoscape 

Network visualization and analysis. Over 200 plugins [60]

http://www.cytoscape.org/

Dizzy

Chemical kinetics stochastic simulation software

http://magnet.systemsbiology.net/software/Dizzy/

DyCoNet

Gephi plugin that can be used to identify dynamic communities in networks

https://github.com/juliemkauffman/DyCoNet

GENeVis 

Network and pathway visualization

http://tinyurl.com/genevis/

GEPHI 

Interactive visualization and exploration for any network and complex system, dynamic and hierarchical graph.

https://gephi.org

Igraph

Collection of network analysis tools with the emphasis on efficiency, portability and ease of use

http://igraph.sourceforge.net

Medusa

Semantic and multi-edged simple networks

https://sites.google.com/site/medusa3visualization/

NAViGaTOR

Visualizing and analyzing protein-protein interaction networks

http://tinyurl.com/navigator1/

N-Browse

Interactive graphical browser for biological networks

http://www.gnetbrowse.org/

NeAT

Topological and clustering analysis of networks

http://rsat.ulb.ac.be/neat/

Ondex 

Data integration and visualization of large networks

http://www.ondex.org/

Osprey

Visualization and annotation of biological networks

http://biodata.mshri.on.ca/osprey/servlet/Index

Pajek 

Analysis and visualization of large networks and social network analysis

http://vlado.fmf.uni-lj.si/pub/networks/pajek/

PathwayAssist 

Navigation and analysis of biological pathways, gene regulation networks and protein interaction maps.

http://www.ariadnegenomics.com/downloads/

PIVOT 

Layout algorithms for visualizing protein interactions and families

http://acgt.cs.tau.ac.il/pivot/

ProCope 

Prediction and evaluation of protein complexes from purification data experiments

http://www.bio.ifi.lmu.de/Complexes/ProCope/

ProViz 

Visualization and exploration of interaction networks. Gene Ontology and PSI-MI formats supported

http://cbi.labri.fr/eng/proviz.htm

SpectralNET 

Network analysis and visualizations. Scatter plots and dimensionality reduction algorithms

https://www.broadinstitute.org/software/spectralnet

Tulip 

Enables the development of algorithms, visual encodings, interaction techniques, data models and domain-specific visualizations

http://tulip.labri.fr/TulipDrupal/

VANESA 

Automatic reconstruction and analysis of biological networks and Petri nets based on life-science database information

http://agbi.techfak.uni-bielefeld.de/vanesa/

VANTED 

Network reconstruction, data visualization, integration of various data types, network simulation

http://tinyurl.com/vanted/

yEd

Creation of diagrams manually and import external data

http://tinyurl.com/yEdGraph/

Web tools for network analysis

APID 

Unified protein-protein interactions from BIND, BioGRID, DIP, HPRD, IntAct and MINT

http://bioinfow.dep.usal.es/apid/

Arcadia 

Translates text-based descriptions of biological networks (SBML files) into standardized diagrams (Systems Biology Graphical Notation Process Description maps)

http://arcadiapathways.sourceforge.net/

AVIS 

Viewer for signaling networks

http://actin.pharm.mssm.edu/AVIS2

bioPIXIE 

Discovery of biological networks from diverse functional genomic data

http://pixie.princeton.edu/pixie

CellPublisher

Interactive representations of biochemical processes

http://cellpublisher.gobics.de/

Graphle

Distributed network exploration and visualization of interactive large, dense graphs

http://tinyurl.com/graphle/

GraphWeb 

Web server for graph-based analysis of biological networks

http://biit.cs.ut.ee/graphweb/

Hubba

Web-based service to explore the essential nodes in a network

http://hub.iis.sinica.edu.tw/Hubba

NetworkBLAST 

Analysis of protein interaction networks across species to infer protein complexes that are conserved in evolution

http://www.cs.tau.ac.il/~bnet/networkblast.htm

Pathview 

Tool set for pathway-based data integration and visualization

http://Pathview.r-forge.r-project.org/

PINA 

Integrated platform for protein interaction network construction, filtering, analysis, visualization and management

http://cbg.garvan.unsw.edu.au/pina/home.do

ReMatch 

Web-based tool for integration of user-given stoichiometric metabolic models into a database collected from public data sources

http://www.cs.helsinki.fi/group/sysfys/software/rematch/

SNOW 

Gene mapping on a reference or human protein-protein interaction network that SNOW hosts

http://snow.bioinfo.cipf.es

STITCH 

Resource to explore known and predicted interactions of chemicals and proteins

http://stitch.embl.de/

STRING

Protein interaction networks and integration of data such as genomic context, high-throughput experiments, conserved coexpression and previous knowledge derived from the literature

http://string-db.org

TVNViewer 

An interactive visualization tool for exploring networks that change over time or space

http://www.sailing.cs.cmu.edu/main/?page_id=545

tYNA 

System for managing, comparing and mining multiple networks

http://tyna.gersteinlab.org/tyna/

VisANT 

Visualization, mining, analysis and modeling of biological networks, metabolic networks and ecosystems

http://visant.bu.edu/

https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4724-8

Address of the bookmark: https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4724-8

Computational methods used by the Shasta assembler include:

• Using a run-length representation of the read sequence. This makes the assembly process more resilient to errors in homopolymer repeat counts, which are the most common type of errors in Oxford Nanopore reads.
• Using in some phases of the computation a representation of the read sequence based on markers, a fixed subset of short k-mers (k ≈ 10).

More at https://chanzuckerberg.github.io/shasta/index.html

Address of the bookmark: https://github.com/chanzuckerberg/shasta

During the first phase of ENCODE, published in 2007, microarray-based technologies were used to detect regions associated with transcription factors, certain histone modifications and open chromatin within a pre-specified 1% of the human genome.

ENCODE’s second phase saw a switch to sequencing-based technologies, the addition of new assay types and the analysis of functional elements genome-wide, described in a collection of research articles in 2012.

The Encyclopedia paper of ENCODE 3, published in Nature, gives an overview of the various assays that were performed in human and mouse cell lines and tissues and describes a Registry of human and mouse candidate cis-regulatory elements (cCREs).

More at https://www.nature.com/immersive/d42859-020-00027-2/index.html

Reference:

https://www.pacb.com/blog/young-investigators-share-stellar-science-career-advice-and-bioinformatics-tools-at-smrt-leiden-2021/

Address of the bookmark: https://github.com/rrwick/Unicycler

Again, running spades.py will show you the options:

spades.py

This produces:

SPAdes genome assembler v3.10.1

Usage: /usr/local/SPAdes-3.10.1-Linux/bin/spades.py [options] -o <output_dir>

Basic options:
-o      <output_dir>    directory to store all the resulting files (required)
--sc                    this flag is required for MDA (single-cell) data
--meta                  this flag is required for metagenomic sample data
--rna                   this flag is required for RNA-Seq data
--plasmid               runs plasmidSPAdes pipeline for plasmid detection
--iontorrent            this flag is required for IonTorrent data
--test                  runs SPAdes on toy dataset
-h/--help               prints this usage message
-v/--version            prints version

Input data:
--12    <filename>      file with interlaced forward and reverse paired-end reads
-1      <filename>      file with forward paired-end reads
-2      <filename>      file with reverse paired-end reads
-s      <filename>      file with unpaired reads
--pe<#>-12      <filename>      file with interlaced reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-1       <filename>      file with forward reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-2       <filename>      file with reverse reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-s       <filename>      file with unpaired reads for paired-end library number <#> (<#> = 1,2,..,9)
--pe<#>-<or>    orientation of reads for paired-end library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--s<#>          <filename>      file with unpaired reads for single reads library number <#> (<#> = 1,2,..,9)
--mp<#>-12      <filename>      file with interlaced reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-1       <filename>      file with forward reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-2       <filename>      file with reverse reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-s       <filename>      file with unpaired reads for mate-pair library number <#> (<#> = 1,2,..,9)
--mp<#>-<or>    orientation of reads for mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--hqmp<#>-12    <filename>      file with interlaced reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-1     <filename>      file with forward reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-2     <filename>      file with reverse reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-s     <filename>      file with unpaired reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9)
--hqmp<#>-<or>  orientation of reads for high-quality mate-pair library number <#> (<#> = 1,2,..,9; <or> = fr, rf, ff)
--nxmate<#>-1   <filename>      file with forward reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--nxmate<#>-2   <filename>      file with reverse reads for Lucigen NxMate library number <#> (<#> = 1,2,..,9)
--sanger        <filename>      file with Sanger reads
--pacbio        <filename>      file with PacBio reads
--nanopore      <filename>      file with Nanopore reads
--tslr  <filename>      file with TSLR-contigs
--trusted-contigs       <filename>      file with trusted contigs
--untrusted-contigs     <filename>      file with untrusted contigs

Pipeline options:
--only-error-correction runs only read error correction (without assembling)
--only-assembler        runs only assembling (without read error correction)
--careful               tries to reduce number of mismatches and short indels
--continue              continue run from the last available check-point
--restart-from  <cp>    restart run with updated options and from the specified check-point ('ec', 'as', 'k<int>', 'mc')
--disable-gzip-output   forces error correction not to compress the corrected reads
--disable-rr            disables repeat resolution stage of assembling

Advanced options:
--dataset       <filename>      file with dataset description in YAML format
-t/--threads    <int>           number of threads
                                [default: 16]
-m/--memory     <int>           RAM limit for SPAdes in Gb (terminates if exceeded)
                                [default: 250]
--tmp-dir       <dirname>       directory for temporary files
                                [default: <output_dir>/tmp]
-k              <int,int,...>   comma-separated list of k-mer sizes (must be odd and
                                less than 128) [default: 'auto']
--cov-cutoff    <float>         coverage cutoff value (a positive float number, or 'auto', or 'off') [default: 'off']
--phred-offset  <33 or 64>      PHRED quality offset in the input reads (33 or 64)
                                [default: auto-detect]

As you can see this is also a “pipeline” of tools that can be switched on or off. SPAdes takes quite a long time, so for the purposes of this practical, something like this may suffice:

spades.py -t 4 \
          -m 32 \
          -k 31,51,71 \
          --only-assembler \
          -1 miseq.1.fastq -2 miseq.2.fastq \
          --nanopore minion.fastq \
          -o hybrid_assembly

In turn, these parameters mean

• use 4 threads
• max memory is 32Gb
• use 3 kmer values to build the de bruijn graph(s) - 31, 51 and 71
• only run the assembler, not the correction algorithm (for speed)
• read 1 and read 2 of the MiSeq data
• the nanopore data
• put the output in folder “hybrid_assembly”