Diagnostic and Investigative Applications of NGS

1. Comprehensive Pathogen Detection (Metagenomics)

In some diagnostic cases, traditional testing fails to identify a causative agent despite strong evidence of infectious disease. In such cases, shotgun metagenomic sequencing can be used to detect nucleic acids from microorganisms present in a sample, including viruses, bacteria, fungi, and parasites.

Sequencing data are analyzed using curated reference databases to identify organisms most likely to explain the observed disease phenotype.

This approach is particularly useful for:

• Culture-negative infections
• Syndromic disease investigations
• Novel or unexpected pathogens
• Co-infections and complex disease etiology

2. High-Resolution Pathogen Identification

In some cases, organisms are successfully isolated from clinical specimens, but traditional phenotypic tests or targeted molecular assays cannot resolve identity with confidence. Whole-genome sequencing offers highly sensitive and specific taxonomic classification and can often resolve identity beyond the species level to subspecies, lineage, or strain.

This level of resolution is critical for:

• Differentiating closely related species with overlapping phenotypes
• Confirming identity of atypical or variant strains
• Resolving discrepancies between phenotypic and molecular test results
• Supporting regulatory or reportable disease investigations

Strain-level identification also provides a foundation for downstream analyses such as virulence profiling, antimicrobial resistance detection, and comparative genomics.

3. Pathogenomics: Linking Genotype to Disease Potential

Pathogenomics focuses on understanding how genetic features of a microorganism relate to pathogenicity. By analyzing whole-genome sequencing data, we can identify genes and genomic regions associated with virulence, host adaptation, immune evasion, and antimicrobial resistance.

This information supports:

• Interpretation of clinical severity or unusual disease presentations
• Differentiation of commensal vs. opportunistic vs. highly pathogenic strains
• Risk assessment for zoonotic transmission
• Integration with clinical and epidemiological metadata

4. Epidemiological Tracing and Strain Relatedness

One of the most powerful applications of microbial whole-genome sequencing is the ability to compare isolates at genome-wide resolution. By quantifying genetic similarity, we can assess whether isolates are closely related or distinct, which supports outbreak investigations, biosecurity decisions, and surveillance.

This analysis is useful for questions such as:

• Is a pathogen persisting within a facility or being repeatedly introduced?
• Are cases from different farms epidemiologically linked?
• Are isolates consistent with vaccine-derived strains or circulating field strains?
• Is an outbreak driven by clonal expansion or multiple introductions?