A breakthrough technology that can identify any virus—known or unknown—is transforming how we detect and respond to infectious diseases.
It started with sinusitis. Dev Mittar, a scientist with a background in microbiology, experienced nasal congestion, fever, and a high heart rate. His doctor assumed a bacterial infection and prescribed antibiotics without running any tests. When they didn't work, a specialist declared it a viral infection that would resolve on its own—but it didn't. Over two months, Mittar endured multiple courses of unnecessary antibiotics as doctors scrambled to identify the cause of his illness. "A single test to rule out infection could have saved me the unnecessary doses of oral antibiotics and IV antibiotics," he recalled. "I had at least 3-4 antibiotic courses, which were totally unnecessary" 5 .
Targeted tests look for specific known pathogens but miss unexpected or novel viruses.
Sequencing all genetic material in a sample enables detection of any virus present.
This diagnostic frustration isn't unique. In clinics and laboratories worldwide, doctors and scientists face similar challenges when confronting mysterious infections. Traditional tests look for specific, known pathogens—but what if the culprit is completely new or unexpected? This critical gap in our diagnostic capabilities has fueled the development of a revolutionary approach: agnostic sequencing for viral pathogen detection. Unlike traditional tests that search for specific suspects, this technology can identify any virus present in a sample, even ones we've never seen before 1 5 .
For decades, viral diagnostics have relied on targeted approaches. Techniques like PCR (polymerase chain reaction) work like a specific key designed to fit a particular lock—they can only detect pathogens they've been specifically designed to find. While these methods are excellent for identifying known viruses like COVID-19 or influenza, they come with a significant limitation: they cannot detect unknown or unexpected pathogens 1 3 .
"The 2019 coronavirus disease (COVID-19) pandemic has emphasized the need for the development, translation, and deployment of pathogen-agnostic molecular diagnostic techniques for use in clinical and public health laboratories," note researchers in Clinical Microbiology Reviews. "Despite recent advancements in molecular diagnostic and sequencing techniques for emerging pathogens, most techniques are targeted in what they detect, relying on a priori knowledge of a pathogen's genome" 1 .
This limitation has real-world consequences. During the early stages of outbreaks, when a pathogen is unknown, health authorities must scramble to develop new tests from scratch, losing valuable time in containing the spread. Agnostic sequencing offers a paradigm shift—instead of multiple specific tests, a single universal test that can identify any virus, known or unknown 5 .
Agnostic sequencing, formally known as metagenomic next-generation sequencing (mNGS), operates on a fundamentally different principle than targeted tests. Rather than hunting for specific pathogens, it sequences all detectable nucleic acids (DNA and RNA) in a sample, then uses sophisticated bioinformatics to identify what's present 1 .
All DNA and RNA is extracted from the sample, whether from a patient, environmental source, or animal.
The genetic material is processed and prepared for sequencing using specialized kits that fragment the nucleic acids and add sequencing adapters 4 .
Next-generation sequencers read all the genetic material in the sample in a massive, parallel process 2 .
Sophisticated software compares the sequenced fragments against extensive databases of known microbial genomes to identify pathogens 3 .
Scientists determine which identified organisms might be causing disease versus incidental findings.
How does agnostic sequencing perform in real-world scenarios? Researchers at Johns Hopkins Applied Physics Laboratory (APL) conducted a comprehensive assessment for the Department of Defense to answer a critical question: when should military applications use traditional PCR versus agnostic sequencing 3 ?
The APL team designed a scenario-based test simulating a febrile patient infected with an unknown biothreat agent. They spiked human blood and serum with four different Biosafety Level 2 or 3 agents representing different types of pathogens:
These pathogens were tested at concentrations corresponding to human clinical disease levels reported in the literature. Each sample was processed using three different viral-enrichment sequencing protocols and compared to agent-specific qPCR assays 3 .
The study yielded nuanced insights into the capabilities of agnostic sequencing:
| Pathogen Type | Example | Agnostic Sequencing Detection | PCR Detection |
|---|---|---|---|
| Double-stranded DNA virus | Vaccinia virus | Successful | Successful |
| Single-stranded RNA virus | VEEV, Hantavirus | Successful | Successful |
| Bacterial agent | Bacillus anthracis | Successful | Successful |
| Unknown novel pathogen | No prior knowledge | Possible | Impossible |
| Very low abundance targets | Early infection | Variable, may miss | More sensitive |
The research demonstrated that "after direct spiking of human blood and serum with biothreat simulants, agnostic sequencing can achieve detection." However, for known agents, PCR still maintained advantages in "speed, cost, scale, and reliability for military applications" 3 .
This crucial finding helps define the appropriate use cases for each technology: PCR remains the best choice when a specific pathogen is suspected, while agnostic sequencing becomes invaluable when facing completely unknown threats or when traditional tests have failed to identify a cause 3 .
Another study comparing different sequencing platforms for virus discovery found that Oxford Nanopore Technology (ONT) performed similarly to Ion Torrent sequencing in identifying pathogens in a prepared library, with ONT offering advantages in turnaround time and ease of use 6 .
Conducting agnostic sequencing requires specialized reagents and equipment. Here are the key components researchers use in their viral detection workflows:
| Tool Category | Specific Examples | Function in Workflow |
|---|---|---|
| Library Prep Kits | NEXTFLEX® kits, Illumina sequencing kits | Convert raw DNA/RNA into format suitable for sequencing by fragmenting nucleic acids, repairing ends, and adding adapters 4 2 |
| Nucleic Acid Extraction Reagents | Various DNA/RNA extraction kits | Isolate and purify genetic material from clinical samples while removing inhibitors |
| Sequence Enrichment Methods | SISPA, SISPA-RACE, Hybrid oligonucleotide enrichment | Enhance detection of viral sequences relative to background material 3 |
| Sequencing Platforms | Illumina, Ion Torrent, Oxford Nanopore | Perform the actual reading of genetic code in a massively parallel manner 2 6 |
| Bioinformatics Tools | EDGE Bioinformatics, custom pipelines | Analyze sequencing data, compare to databases, and identify pathogens 3 |
Each component plays a critical role in the process. For instance, library preparation kits have been developed for full compatibility with leading sequencing platforms, ensuring they integrate smoothly into laboratory workflows. Specialized enrichment methods like SISPA (sequence-independent, single-primer amplification) employ techniques that allow viral amplification without prior knowledge of the viral genome, making them particularly valuable for unknown pathogen discovery 3 4 .
Despite its promise, agnostic sequencing faces several hurdles before becoming a routine diagnostic tool. The technology remains relatively complex and expensive compared to traditional tests, requires significant expertise to implement, generates massive amounts of data that challenge interpretation, and needs regulatory approval for widespread clinical use 3 5 .
"The cost of sequencing is falling dramatically," notes Mittar. "Think of sequencing technology 25 years ago. Probably in 2001 or 2002, sequencing the human genome would cost like 100 million dollars, the cost of a Boeing 737" 5 .
The future may see agnostic sequencing combined with other advanced technologies. For instance, when coupled with metatranscriptomics, it could provide information about a patient's immune profile alongside pathogen detection, potentially enabling more personalized treatment approaches 5 .
| Parameter | Traditional PCR | Rapid Antigen Tests | Agnostic Sequencing |
|---|---|---|---|
| Detection Scope | Known, targeted pathogens only | Known, targeted pathogens only | All detectable pathogens |
| Novel Pathogen Detection | Not possible | Not possible | Possible |
| Speed | Minutes to hours | Minutes | Hours to days |
| Cost | Low | Very low | Currently high |
| Expertise Required | Moderate | Low | High |
| Data Richness | Low (presence/absence) | Low (presence/absence) | High (genetic information) |
As research progresses, the goal is to develop complete "sample-to-answer agnostic diagnostic solutions" that can be deployed rapidly at the first sign of an outbreak, ensuring an effective response to emerging viral threats 1 5 .
Agnostic sequencing represents a fundamental shift in our approach to microbial threats. By moving from targeted hunting to universal surveillance, this technology offers our best defense against the unknown pathogens that will inevitably emerge in our interconnected world.
Traditional diagnostics could only identify known pathogens, leaving novel threats undetected until outbreaks were established.
Agnostic sequencing enables detection of any virus but remains complex and expensive for routine use.
As costs decrease and technology improves, agnostic sequencing may become a standard tool for clinical diagnostics and outbreak response.
While challenges remain, the progress has been remarkable. From the days of being unable to detect novel viruses until they had already spread widely, we're approaching an era where any viral pathogen can be identified quickly, even during the earliest stages of an outbreak. As this technology becomes more accessible and refined, it may transform from a specialized tool into a routine part of clinical care—potentially saving patients like Dev Mittar from months of diagnostic uncertainty and unnecessary treatments.
In the continuous arms race between humans and microbes, agnostic sequencing provides a powerful new weapon—one that doesn't just help us fight the pathogens we know, but prepares us for the ones we have yet to meet.
"The concept is simple: you take a clinical sample, run one test, and you can identify any pathogen—be it virus, bacteria, fungi, even parasites—no matter existing or new." — Dev Mittar, Ph.D. 5