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Introducing ONETest™ PathoGenome
Microbial Core-Genome Enrichment for Comprehensive Profiling

Undiagnosed infections including hospital-acquired,  respiratory, blood and CNS  infections continue to challenge healthcare systems worldwide. At the heart of this problem lies a persistent diagnostic gap: how to identify diverse pathogens rapidly, accurately, and comprehensively from complex clinical samples.

ONETest™ PathoGenome (PG) is a next-generation solution built for this purpose. It is a metagenomics assay that uses proprietary QuantumProbes™ to enrich core-genome loci across a broad spectrum of microbial families—including bacteria, fungi, and DNA viruses. Unlike traditional amplicon or probe panels, ONETest™ PG does not rely on rigid detection panels or predefined targets. Instead, it captures the evolutionary backbone of microbial genomes: the core genes shared by nearly all strains within a taxonomic group.

This approach offers two transformative advantages:

1. Lineage-Wide Coverage: QuantumProbes™ are engineered to tolerate up to ~20% sequence divergence, allowing the assay to enrich both well-characterized pathogens and their phylogenetically related, divergent counterparts.
2. Species-Level Resolution: By targeting polymorphic but conserved core loci, ONETest™ PG achieves high signal-to-noise detection that supports confident taxonomic placement, even in commensal-rich matrices such as BAL or tracheal aspirates.

Assay Advantages for Hospital and Research Use:

• Core-Genome Coverage: Captures loci from 254 microbial species spanning 50 families commonly investigated in respiratory infection research
• Designed for Evolutionary Capture: QuantumProbes™ are engineered to enrich conserved core-genome loci across species, genus, family, and phylum levels—enabling detection across evolutionary distances by design, not by chance
• High-Resolution Profiling: Enables species-level characterization of microbial DNA sequences and related taxa
• Captures Entire Lineages: Unlike assays relying on predefined panels or short amplicons, ONETest™ PG can capture entire microbial lineages, positioning it as a new class of targeted metagenomic assay
• Novel Pathogen Identification: Capable of identifying emerging or uncharacterized pathogens that fall within covered taxonomic families
• Designed for Complex Matrixes: Optimized for use with complex matrixes such as BAL and tracheal aspirates containing high host and commensal DNA background
• Proprietary Probe Design: QuantumProbes™ tolerate sequence divergence, allowing capture of both reference and divergent genomic regions
• Automated Workflow: Fully automated protocol on Hamilton STAR liquid handler
• Integrated Bioinformatics: FusionCloud™ enables secure, end-to-end genomic data analysis—automating everything from demultiplexing to taxonomic assignment, signal strength classification, and reporting. It transforms sequencing data into actionable microbial profiles without requiring local infrastructure.

Workflow Summary:

1. Sample Collection: BAL or tracheal aspirate (700–2000 µL)
2. DNA Extraction: MagMax™ Microbiome Ultra Kit (ThermoFisher)
3. Library Prep: Enzymatic fragmentation and dual indexing (Illumina-compatible)
4. Target Enrichment: QuantumProbes™, magnetic bead pull-down
5. Sequencing: Illumina 2×150 bp
6. Data Analysis: Via FusionCloud™ with core-genome alignment, taxonomic placement, and quantitative metrics

FusionCloud™ Bioinformatics Platform
ONETest™ PG’s performance is powered not just by its core-genome capture chemistry—but also by its integrated informatics engine. The FusionCloud™ platform is a secure, cloud-based environment that automates all downstream processing steps:

• Host read removal and quality filtering
• Alignment to curated reference databases
• Taxonomic classification via k-mer and alignment-based methods
• Core-genome reconstruction and variant calling
• Automated signal categorization (Strong, Moderate, Weak, Background)
• Downloadable, publication-ready reports and visualizations

FusionCloud™ is optimized for high-host-content samples like BAL, and supports cross-sample comparisons for cohort studies or infection surveillance programs or part of analysis portal in an LDT environment.

Validation Summary:

• Limit of detection testing conducted with twelve representative microbial species
• 64 clinical BAL samples tested; 35 microbial taxa observed representing 17 microbial families
• Captured core-genome loci shared with 150 of the 254 ONETest™ panel species (~59% target coverage)
• High concordance with whole metagenome sequencing for species richness (R = 0.79, p < 0.001)
• Validation results support ONETest™ PG’s design-driven ability to capture core-genome regions across a wide phylogenetic range

Deliverables:

• Mapped read counts and FPKM values
• Core-genome assemblies and taxonomic inference
• Signal strength categorization: Strong, Moderate, Weak, or Background
• Downloadable visualizations and structured reports via FusionCloud™

Intended Users:

• Hospital microbiology and infectious disease labs conducting research or LDT validation
• Academic research teams investigating respiratory microbiomes or healthcare-associated infections
• Translational collaborations exploring antimicrobial resistance and emerging pathogen surveillance

Regulatory Status: RUO – Not for diagnostic use

* preliminary pathogen identification report will be provided at the end of 30 cycles of DNA sequencing.
**Up to 24 samples in one desktop sequencer run.