16S rRNA and ITS amplicon sequencing identifies which microorganisms are present in a sample — and at roughly what proportion — without sequencing the whole metagenome. It's the right starting point for any project where the budget is tight and/ or compositional data at genus level is sufficient.
Amplicon sequencing works by targeting a short stretch of DNA that nearly all bacteria (16S rRNA gene) or fungi (ITS region) share, but in slightly different forms between species. By amplifying and sequencing this marker across thousands of organisms in a mixed sample, you can identify which groups are present and in roughly what proportion — without sequencing the entire metagenome. It is substantially cheaper than shotgun metagenomics, scales well to hundreds of samples in a single run, and produces results that are straightforward to interpret with established pipelines (QIIME2, DADA2, mothur).
The limitations are equally real, and understanding them determines whether amplicon sequencing is the right tool for your question. Resolution is the primary one: 16S V3-V4 amplicons typically allow classification to genus level reliably, and to species in some genera — but most amplicon studies cannot distinguish closely related species, and strain-level identification is generally not possible. This matters a great deal if your question requires species-level precision (distinguishing Bifidobacterium longum from B. breve, for example), but not at all if you are characterising broad community-level shifts across a clinical cohort. Know which category your question falls into before committing a sequencing approach.
Amplification bias is the second limitation worth understanding explicitly. PCR primers don’t amplify all organisms equally — some taxa are consistently under- or over-represented depending on primer choice and amplification conditions. The most commonly used V4 primers (515F/806R) cover the vast majority of bacterial 16S diversity but miss some organisms entirely (some Bifidobacterium strains, certain Archaea, some Firmicutes). For most human gut or environmental microbiome studies this is an acceptable tradeoff. For clinical applications or studies with known biases in your target community, primer choice deserves careful consideration.
Fungi require a different marker — the ITS (internal transcribed spacer) region rather than 16S. ITS1 and ITS2 primers both have broad coverage but different biases; ITS2 generally performs better for environmental samples, ITS1 for some clinical fungi. For samples expected to contain both bacteria and fungi (most human gut and environmental samples), separate 16S and ITS amplifications are required — they cannot be co-amplified reliably. We can run both in the same project with coordinated sampling.
If you need functional data (metabolic pathways, resistance genes, virulence factors), species- or strain-level resolution, detection of viruses or non-16S-containing organisms, or a truly unbiased community profile, amplicon sequencing will not give you what you need. We will tell you this directly during consultation rather than take a project that won’t answer your question. The difference in cost between amplicon and metagenomics is substantial, but cost efficiency only matters if the data is actually fit for purpose.
Amplicon sequencing is a composition-only tool. It cannot tell you what the community is doing — only who is there and in what proportion. For functional questions, clinical applications requiring species-level resolution, or any project where you suspect what you're looking for isn't a bacterium or fungus, discuss your question with us before committing a method.
Clinical trials, epidemiological studies, intervention studies — anywhere the sample count is in the hundreds or thousands and the question is compositional. The cost per sample makes cohort sizes that would be prohibitive with metagenomics entirely practical. For a standard gut microbiome study with a compositional endpoint, amplicon sequencing at 50,000–100,000 reads per sample provides more than sufficient depth to capture the dominant community structure and detect meaningful between-group differences.
Tracking community dynamics over time — across treatment cycles, developmental stages, environmental perturbations, or fermentation processes. The high sample throughput and low per-sample cost of amplicon sequencing makes dense longitudinal sampling designs (10+ timepoints per subject, 50+ subjects) financially realistic. Paired with a consistent protocol across all timepoints, amplicon sequencing produces comparable data for tracking compositional shifts over time.
Monitoring microbial community composition in fermentation, food production, brewing, dairy, and agricultural processes — where the question is whether the intended community is present in expected proportions, or whether contaminants or off-target organisms are emerging. Amplicon sequencing is well-suited to routine QC: it is fast, scalable, and produces actionable compositional data without the analytical complexity of full metagenomics.
Soil, water, sediment, air filter — any environment where you need to characterise and track microbial community composition over time or across sites. Amplicon sequencing is the most widely used method for environmental microbiome surveys, with extensive reference databases and standardised analytical pipelines. For biodiversity assessments, regulatory monitoring, or environmental impact studies where composition rather than function is the question, it is often the most pragmatic choice.
Amplicon sequencing can serve as a cost-efficient first step before committing to full metagenomics on a large cohort: confirming that communities vary across conditions as expected, identifying which samples are low biomass, and checking that collection protocols are producing consistent results. Running a small amplicon pilot (20–40 samples) before a large metagenomics project is a straightforward way to validate experimental design at low cost.
Specifications for standard 16S V3-V4 and ITS1/ITS2 amplicon projects. Contact us about other target regions, custom primer sets, or paired 16S + ITS projects.
Four stages from first contact to ASV tables and taxonomy data. Amplicon sequencing is one of our most streamlined workflows — most projects are in sequencing within three days of sample receipt.
We confirm your target (16S, ITS, or both), primer set, expected sample count, and any cohort-level requirements. We'll also discuss negative control strategy — this is especially important for low-biomass samples where environmental contamination in reagents can dominate signal — and check that your collection protocol is compatible with downstream PCR amplification.
Primer selection matters more for some communities than others — we'll tell you honestly if a different primer set would serve your question better.
Written proposal covering target region, primer set, depth per sample, controls, agreed bioinformatics add-ons, turnaround, and pricing. For large cohorts, we include batch processing plan and multiplexing strategy so you know exactly how your samples are distributed across runs.
Batch effects are the most common source of technical variation in large amplicon studies. We plan run layouts to minimise them.
We confirm receipt of samples or pre-extracted DNA, check concentration and quality (Qubit, NanoDrop), and flag any samples below threshold before PCR begins. For soil and environmental samples, we test for PCR inhibition before library preparation. Failed or inhibited samples are reported immediately — not after a full library prep run has been wasted.
For pre-extracted DNA: note which kit was used. Kit-dependent biases are real and we account for them in QC interpretation.
Two-step PCR protocol: first PCR amplifies the target region; second PCR adds sample-specific indices for multiplexing. Library normalisation, pooling, and quality check (TapeStation) before sequencing. Positive and negative controls processed alongside every plate. Illumina sequencing; demultiplexing and quality filtering before delivery.
Two-step PCR and per-plate negative controls are standard best practice for minimising cross-contamination artefacts in multiplexed amplicon runs. Contact us if you are interested in One-step PCR.
Both are valid choices and both are widely used. V3-V4 (341F/805R, producing ~460 bp amplicons) covers a larger region of the 16S gene, providing slightly better taxonomic resolution for some genera, and is the standard in many European research networks. V4 (515F/806R, ~253 bp amplicons) has broader primer coverage across diverse bacterial phyla, pairs well with 2×250 bp sequencing runs, and is the primer set used by the Earth Microbiome Project — giving it the largest reference database of any single 16S primer set. For most human gut microbiome studies, both perform similarly. For environmental samples with unusual community compositions, V4’s broader coverage is often preferable. We’ll recommend based on your organism context and whether cross-study comparability with a specific public dataset matters to your analysis.
Several things. First, DNA extraction from soil is harder than from biological samples: soil contains humic acids and other compounds that inhibit PCR. Standard extraction kits designed for stool or clinical samples often fail on soil, and inhibitor removal steps (PowerSoil + PowerClean columns, or CTAB-based protocols) are important. We can handle soil extraction if you prefer not to — tell us the soil type and expected community composition. Second, low-biomass environments (clean water, air filters, some desert soils) require very careful contamination control in extraction and PCR. Third, for aquatic samples, the filtration membrane material affects extraction efficiency — use 0.22 µm polycarbonate or PVDF, not cellulose acetate. Contact us before collection if you are working with an unusual environmental matrix.
The standard answer — 30,000 reads per sample — is adequate for most gut microbiome studies with relatively simple community structures and clear group-level differences. The situations where it’s not adequate: low-diversity communities where very rare organisms matter (you need more reads to detect organisms present at <0.1% relative abundance), high-diversity environmental samples (soil, marine sediment), and projects focused on differential abundance of specific low-abundance taxa. Rarefaction curves are the definitive answer to whether a given depth is sufficient for a given sample: you sequence to a depth where the curve levels off. For first-time projects in a new environment, we often recommend sequencing a small pilot at higher depth to establish what’s sufficient before scaling. Under-sequencing is cheaper to fix than wrong primer selection, but still worth getting right upfront.
No. Amplicon sequencing targets a marker gene for community identification; it tells you nothing about what resistance genes are present, what metabolic functions the community carries, or what the organisms are actually doing. For AMR questions — whether resistance genes are present, which organisms carry them, what mobile elements are involved — you need shotgun metagenomics. This is one of the clearest and most important distinctions between the two methods, and it’s a common source of confusion in study design. A community that looks similar at the 16S level can have dramatically different resistomes.
Tell us about your sample type, target community (bacteria, fungi, or both), expected cohort size, and biological question. We'll respond as fast as we can — or arrange a call if the study design needs more discussion.