Same Sample, Different Story: Why qPCR and Shotgun Microbiome Tests Don’t Always Match
Recently, a client asked me why Citrobacter and Pseudomonas, flagged as high on a microbiome test earlier in the year, were nowhere to be seen in her new results. The first was a GI-MAP qPCR test. The second was a CoBiome shotgun metagenomic panel.
Same gut. Different story.
This isn’t unusual - in fact, I see this kind of discrepancy all the time. And it’s not just GI-MAP. The same applies to other qPCR-based microbiome panels like those offered by Nutripath (AUS), US BioTek (USA), and several others.
So what’s going on?
Different Tests, Different Technologies
At the heart of it, these tests are asking different questions using different tools.
qPCR (quantitative polymerase chain reaction) tests like GI-MAP target a short list of specific microbes using pre-designed DNA probes. If a match is found, it’s reported - sometimes even at trace levels.
Shotgun metagenomics, on the other hand, sequences all microbial DNA in the sample. This allows for detection of hundreds to thousands of species (and sometimes even strains), offering a much broader view of your microbiome.
One isn’t necessarily better than the other - they’re simply built for different purposes. But understanding those differences is key to interpreting results in context.
The qPCR Approach: Focused but Limited
qPCR is like shining a spotlight in the dark. It’s precise - but only within its limited field of view.
The lab chooses which microbes to test for, based on what they consider clinically relevant or commonly found. For example, GI-MAP’s panel includes some commensals, opportunists, and pathogens - but only those the lab has included in its internal list. If you have a microbe that isn’t on their radar, it simply won’t show up.
Even more critically, qPCR tests can sometimes misclassify microbes due to shared genetic regions between related species. DNA primers used to identify microbes may also bind to the wrong organism - especially in genera like Citrobacter, Enterobacter, and Klebsiella, where some genetic regions are highly conserved.
This problem is well documented in microbial sequencing studies. One comparative study showed that primers targeting the 16S rRNA gene - a common approach in qPCR and some sequencing tests - can fail to distinguish between closely related bacterial species.
And because qPCR amplifies all DNA - live or dead - it’s not uncommon for these tests to pick up fragments from transient or inactive microbes, making it hard to judge clinical relevance from detection alone.
Some labs - like Nutripath, Doctor’s Data and Genova Diagnostics - combine qPCR with traditional stool culturing, which can add value by providing antimicrobial susceptibility testing for certain bacteria or yeast. This hybrid approach can offer insight into potential treatment options, particularly for overgrowths like Candida or Klebsiella. But it's important to remember that many gut microbes don’t grow well outside the body, and culturing tends to detect only a narrow range of organisms. So, while this method can complement PCR data, it still reflects just a small portion of the broader microbiome ecosystem.
The Shotgun Metagenomics Approach: Wide and Deep
Shotgun metagenomics takes a completely different approach. Instead of targeting specific species, it sequences millions of DNA fragments from your entire stool sample, then compares them against large reference libraries to identify what’s there - and at what relative abundance.
The benefits?
It captures everything, not just what’s been preselected.
It allows you to see the full ecosystem, including beneficial keystone species and hard-to-grow microbes.
It offers insight into functional potential - such as butyrate production, diversity, and gut resilience.
And it reduces the risk of misidentification, because species-level calls are made from whole genome sequences, not just short genetic markers.
That said, shotgun tests may not list every species either. Most platforms set cut-offs to avoid overwhelming reports with data (e.g. microbes under 0.01% abundance may be filtered out). So if Citrobacter doesn’t appear, it may still be there - just at low levels, or not functionally significant enough to make the summary.
The Problem with Proprietary Panels and Secret Databases
One of the major issues with many qPCR-based labs is lack of transparency.
Companies like GI-MAP, Nutripath, and US BioTek do not disclose:
Their primer sequences (the DNA targets used in their assays),
The validation data for species specificity,
Or the databases used to confirm microbial identity.
This makes it almost impossible for clinicians or researchers to verify the accuracy of results - and raises concerns about false positives, cross-reactivity, and over-reporting of pathobionts that may not be clinically meaningful.
In contrast, shotgun metagenomics labs (e.g. Microba, NirvanaBiome, TinyHealth) typically align their sequencing results to well-maintained public databases like RefSeq, NCBI, or GTDB. These are regularly updated, peer-reviewed, and publicly accessible, which allows for greater consistency and transparency.
Of course, the field isn’t perfect - microbiome reference libraries are still evolving, and some species remain poorly characterised. But the openness of metagenomic methods makes it easier to track, compare, and trust what’s being reported.
Clinical Relevance vs Presence: A Shift in Focus
Another key difference is the philosophy behind each approach.
qPCR panels tend to focus on identifying potential pathogens or "problem microbes," which can lead to overemphasis on pathobionts, even in the absence of symptoms. For example, many of my clients come in concerned about Klebsiella or Candida flagged “high” on a GI-MAP or Microbiome Maping, despite having no digestive symptoms.
Shotgun metagenomic testing, meanwhile, looks at the whole ecosystem - the balance between beneficial and opportunistic microbes, the presence of butyrate producers, mucin degraders, and the diversity patterns that underpin long-term health.
This ecosystem view is often more clinically useful, especially in chronic or subclinical cases.
So, Which Test Is Best?
It depends on your goal.
If you’re investigating acute gastrointestinal infections, qPCR can be helpful - but a standard stool PCR through a medical lab may be more cost-effective and validated.
If you’re exploring gut healing, long-term immune health, or post-antibiotic recovery, shotgun metagenomics provides a richer, more accurate picture.
In my clinic, I’ve moved away from relying on qPCR tests like GI-MAP unless they serve a very specific purpose. I’ve found too many cases where flagged microbes created more confusion than clarity - and where symptoms improved with microbiome restoration, regardless of whether Citrobacter was “high” or not.
Final Thoughts
Not all microbiome tests are created equal. qPCR and shotgun metagenomics are built on different foundations, and their results reflect those differences.
So if your second test looks nothing like your first, don’t assume something’s gone wrong. You may just be looking at your microbiome through a wider lens.
In the end, it’s not just about which microbes show up - it’s about what they’re doing, who they’re surrounded by, and whether your gut has the diversity and function it needs to support your health.
That’s the real story worth listening to.
