16S/18S/ITS Amplicon Sequencing

What is 16S/18S/ITS Amplicon Sequencing?

CD Genomics provides accurate and cost-effective amplicon sequencing for bacteria, archaea, and fungi. Our platform supports diverse applications in environmental science, agriculture, industry, and healthcare.

16S Sequencing
Targets the 16S rRNA gene, present in bacteria and archaea, featuring nine hypervariable regions for detailed microbial identification. By sequencing regions like V3–V4 or V4–V5, we classify microbes from broad groups down to species level. This method is ideal for microbiome analysis, environmental monitoring, and detecting microbial bioindicators.
18S Sequencing
Focuses on the 18S rRNA gene found in eukaryotes including protists, fungi, and plants. Although more conserved than 16S, it effectively distinguishes major eukaryotic groups. Common applications include studying soil and water eukaryotic microbes, biodiversity assessments, and ecological research.
ITS Sequencing
Amplifies the highly variable ITS region, critical for fungal species identification. ITS sequencing excels at differentiating fungi with high precision and is widely used for identifying edible and medicinal fungi, detecting pathogens, and tracing contamination in food and environment.

Targeted Areas and Widely Used Primers for 16S rRNA, 18S rRNA, and ITS Amplification

Target Regions and Common Amplification Primers for 16S rRNA, 18S rRNA, and ITS

16S vs 18S vs ITS: Which Marker Should You Use?

Not sure which marker fits your community and sample type? Use the quick comparison below to avoid choosing a region that misses your target taxa or reduces resolution.

Marker	Best for	Main targets	Typical resolution	Common region choice	Key caveat (pain point)
16S rRNA	Bacterial/archaeal community profiling	Bacteria, Archaea	Genus to species (depends on region)	V3–V4 / V4–V5	Primer/region choice can shift observed composition; species-level may be limited for some taxa
18S rRNA	Eukaryotic microbiome overview	Protists, algae, some fungi/plants	Often higher-level eukaryotic groups	SSU region	Less variable → may under-resolve closely related eukaryotes
ITS (ITS1/ITS2)	Fungal community and species ID	Fungi	Often species-level for fungi	ITS1 or ITS2	Length/GC variability can bias amplification; choose ITS1 vs ITS2 by sample and study goal

Quick pick

If your primary goal is bacteria/archaea diversity → choose 16S.
If you need protists/algae/eukaryotic diversity → choose 18S.
If you need fungal species profiling → choose ITS (ITS1/ITS2).

Why Choose Amplicon Sequencing?

Amplicon sequencing offers a smart, efficient alternative to traditional culturing or full metagenomic sequencing — especially for large-scale or complex microbial studies.

Fast, Accurate Microbial ID: Detects diverse species with higher accuracy than traditional culturing.
High Sensitivity & Throughput: Delivers 50,000+ reads/sample — ideal for low-abundance microbes in complex samples.
Species-Level Resolution: ASV-based analysis (e.g., DADA2) offers finer taxonomic precision than OTU clustering.
Cost-Effective Targeting: Focuses on key gene regions to reduce sequencing and analysis costs.
Rapid Turnaround: Amplicon sequencing enables fast data generation and reporting.

Method Comparison

Method	Turnaround Time	Sensitivity	Cost	Ideal For
Amplicon Sequencing	★★★★☆	0.01%	$	Community diversity
Metagenomic Sequencing	★★☆☆☆	0.1%	$$$$	Functional gene analysis

Amplicon Sequencing Targets & Applications at a Glance

Amplicon sequencing is a powerful tool for profiling microbial communities across diverse sample types and research goals. The table below outlines typical targets and application scenarios for each sequencing type.

Amplicon Region	Target Microorganisms	Key Applications
16S rRNA	Bacteria, Archaea	• Gut microbiome • Pathogen detection • Soil and sediment analysis • Industrial wastewater treatment • Bioindicator screening • Food spoilage monitoring
18S rRNA	Eukaryotes (fungi, algae, protists)	• Aquatic ecosystem dynamics • Marine protist diversity • Soil eukaryotic communities • Airborne microbe tracking • Extreme environment adaptation studies
ITS Region	Fungi	• Fungal taxonomy profiling • Edible and medicinal fungi identification • Plant and animal pathogen detection • Food and pharmaceutical contamination tracing • Agricultural soil fertility studies

16S/18S/ITS Amplicon Sequencing Service Options

We offer three main sequencing options based on research needs:

Standard 16S/18S/ITS Sequencing

Targeted regions | Genus/species-level | Cost-effective

Detailed Parameters ↓

Full-Length 16S/18S/ITS Amplicon Sequencing

Comprehensive profiling | Species/strain-level | High phylogenetic accuracy

Explore Full-Length Service →

Absolute Quantitative 16S/18S/ITS Sequencing

qPCR + Sequencing | Accurate abundance | For quantitative microbiome studies

Explore Absolute Quant Service →

Standard vs Full-Length vs Absolute Quantitative Amplicon Sequencing

Not sure which option fits your study? The key difference is whether you need a cost-efficient relative profile, higher taxonomic resolution, or absolute abundance to track true microbial load changes.

Comparison Table

Option	What it measures	Best for	What you gain	Note / trade-off
Standard (short-region amplicon)	Relative abundance from a commonly used region (e.g., 16S V3–V4; ITS1/ITS2; 18S region)	Large cohorts, routine community comparisons, discovery screening	Fast, scalable profiling + alpha/beta diversity + taxonomy summaries	Resolution depends on region/primers; relative abundance may be influenced by compositional effects
Full-Length	Relative abundance with near full-length marker coverage (long-read strategy)	Higher taxonomic resolution in complex communities (project-dependent)	Improved taxonomy confidence and phylogenetic resolution	More complex sequencing/analysis than short-region approaches
Absolute Quantitative	Absolute abundance (calibrated abundance; copies per unit sample, as applicable) plus relative profiles	Studies where "how much" matters (load shifts, treatment response, biomass change)	Adds quantitative interpretability beyond proportions; supports cross-sample load comparison	Requires appropriate quantification design and QC; results depend on sample matrix and calibration

Quick pick (at a glance)

Choose Standard if you mainly need community structure and between-group comparisons.
Choose Full-Length if you need higher taxonomic resolution for complex samples.
Choose Absolute Quantitative if you need to measure true abundance changes, not only relative proportions (RUO).

End-to-End 16S/18S/ITS Sequencing Workflow

We provide a comprehensive, one-stop service covering the entire workflow—from sample preparation to data analysis—ensuring both data quality and research efficiency.

Workflow for 16S, 18S, and ITS Amplicon Sequencing Services

16S/18S/ITS Amplicon Sequencing Strategy

Sequencing platforms:

Illumina MiSeq™, Illumina NovaSeq 6000™ (PE250)

Effective read length:

200–250 bp after adapter trimming

Target regions:

16S: V1–V2, V1–V3, V3–V4, V3, V4, V4–V5, V5–V6, V6–V8
18S: Standard small subunit region
ITS: ITS1, ITS2
Custom regions: Available upon request

Accepted sample types:

Faeces, soil, water, skin swabs, plant and animal tissues, fermentation broth, extracted DNA, and other environmental or biological materials

What's Included in Our Amplicon Sequencing Bioinformatics Analysis

1. High-Resolution Sequence Processing

DADA2-based ASV generation
Single-nucleotide accuracy to capture real biological variants

2. Taxonomic Annotation

Phylum to species-level classification
Powered by our in-house database for higher accuracy than SILVA/UNITE

3. Microbial Diversity Insights

Alpha diversity (Shannon, Chao1): Understand richness within samples
Beta diversity (PCoA, NMDS): Compare community differences across groups

4. Abundance & Differential Analysis

Visual outputs: bar plots, heatmaps
LEfSe highlights statistically significant microbial changes

5. Optional: Absolute Quantification

Integrate qPCR data to calculate real microbial loads

Bioinformatics Workflow for 16S/18S/ITS Amplicon Sequencing

Key Research Questions Answered

Our microbial amplicon bioinformatics analysis helps you answer critical questions:

Analysis Type	Research Purpose
OTU/ASV Clustering and Taxonomic Annotation	Identifies major microbial taxa present in each sample.
Taxonomic Composition Analysis	Describes microbial distribution across taxonomic ranks such as phylum and genus.
Phylogenetic Analysis	Reveals evolutionary relationships between detected microbial species.
Alpha Diversity Analysis (Within-Sample)	Evaluates the richness and evenness of microbial communities within individual samples.
Beta Diversity Analysis (Between-Sample)	Compares microbial community differences across groups or sample types.
Community Structure Significance Testing	Determines whether inter-group microbial differences are statistically significant.
Differential Abundance Analysis	Detects key microbial taxa that differ significantly between groups or conditions.

16S/18S/ITS Amplicon Sequencing Sample Preparation Guide

Sample Type	Submission Guidelines
Extracted Environmental DNA	• Total ≥ 100 ng • Concentration ≥ 10 ng/μL • Recommended OD260/280 between 1.8–2.0
Genomic DNA	• Total ≥ 100 ng • Concentration ≥ 1 ng/μL • Must be free of RNA and protein contamination
PCR Products	• Total ≥ 3 μg • Concentration ≥ 10 ng/μL • Must be purified; please provide amplicon length and primer sequence information
Raw Samples (e.g., soil, water, sediment)	• Recommended: ≥ 5 g for wet samples, ≥ 2 g for dry samples, or ≥ 5 mL for liquid samples • Ensure sterile packaging and cold chain transport

Shipping Instructions: Samples must be shipped with light protection and under cold conditions (preferably on dry ice or stored at −80°C).

Note: For other sample types, please contact us for a customised protocol.

Why Choose CD Genomics Amplicon Sequencing?

Advanced amplicon sequencing solutions trusted by 500+ global institutes for actionable microbiome insights.

Capture Rare Taxa: Detect microbes as low as 0.01% abundance (≥50,000 reads/sample).
Multi-Region Flexibility: Optimized primers for V3-V4 (bacteria), ITS2 (fungi), and custom regions.
Fast Results: Reports delivered quickly to support your research timeline.
Publication Support: LEfSe, PCoA, and heatmaps formatted for Nature Microbiology guidelines.

16S/18S/ITS Amplicon Seq FAQs

1. How do I choose the right amplicon region (e.g., V3–V4, ITS2) for my sequencing project?

The ideal region depends on your sample type and research goal. Below are common use-case recommendations:

Gut microbiome (human or animal): Use V3–V4 for broad microbial coverage and balanced taxonomic resolution.
Oral or skin microbiota: Try V1–V3 or V1–V2 to better distinguish dominant surface-associated bacteria.
Soil, water, or other environmental samples: Choose V3–V4 or V4–V5, both effective for high-diversity microbial communities.
Fungal profiling: Use ITS2 for general fungal community studies, or ITS1 when analyzing air or plant-associated fungi.
Eukaryotic microbes (e.g., protists): The 18S rRNA SSU region is preferred for soil and aquatic samples.

✅ These are starting points — the best choice ultimately depends on your taxonomic resolution needs and study design. We recommend a brief consultation during project planning to select the most appropriate region and primers.

2. Do I need to include biological replicates? If so, how many?

Yes — replicates are highly recommended, especially for studies involving statistical comparisons or differential abundance analysis.

Minimum suggestion: At least 3 biological replicates per group for standard LEfSe or ANOSIM tests.
For complex or heterogeneous samples: Use 5–6 replicates to improve data reliability and capture community variability.

3. What if my sample quantity or quality doesn't meet requirements?

All incoming samples undergo quality checks. If the DNA is too low in concentration or purity, we'll alert you and suggest alternatives.

Low-yield rescue: In many cases, we can offer optimized protocols for limited samples — contact our support team for options.

4. Can you process difficult or unconventional sample types?

Absolutely. We routinely handle:

Soil, sediment, and sludge
Water (fresh or marine)
Faeces, swabs (skin, nasal), fermentation broths
Other high-contamination or low-biomass samples

⚠️ For extreme environments or low-abundance communities, let us know in advance — we'll tailor the workflow accordingly.

5. Is the bioinformatics analysis customisable?

Yes. Our standard pipeline includes:

Sequence quality control
Taxonomic annotation
α- and β-diversity metrics
Differential abundance (e.g., LEfSe)

For advanced requests — like metabolic pathway prediction, network analysis, or absolute quantification — we offer bespoke analysis packages. Pricing and timelines are quoted case by case.

6. Can I send multiple samples at once? Will they contaminate each other?

Yes, we support high-throughput processing of multiple samples. Each sample is barcoded with a unique identifier.

7. Do I need to supply primers, or are they provided?

We typically provide validated primer sets optimised for commonly used regions like V3–V4 or ITS2. If you need a custom region or primer design, simply share the target and we'll assist with optimization and synthesis.

8: What is the difference between relative and absolute abundance in amplicon sequencing?

Relative abundance shows the proportion of taxa within a sample, while absolute abundance estimates calibrated load (e.g., copies per unit sample) to help distinguish true biomass changes from compositional shifts.