- What is Amplicon Sequencing
- Why Use Amplicon Sequencing
- Choosing the Right Amplicon Sequencing Platform & Read Length
- Our Amplicon Sequencing Process: From Consultation to Reporting
- Research Applications of Amplicon Sequencing
- Amplicon Sequencing Bioinformatics & Data Analysis Services
- Sample Requirements and Quality Guidelines for Amplicon Sequencing
- Why Choose CD Genomics for Amplicon Sequencing?
What is Amplicon Sequencing
Amplicon sequencing is a targeted next-generation sequencing (NGS) approach that uses specifically designed primers to amplify selected genomic regions via PCR, followed by high-throughput sequencing of the amplicons. This technique enables precise detection of genetic variants within defined loci and is widely used in studies of gene mutations, microbial diversity, and biomarker screening.
How It Works
- Target Selection & Primer Design – Primers are designed to amplify specific genomic regions of interest based on research objectives.
- PCR Amplification – The target regions are amplified from sample DNA to generate high-abundance amplicons.
- Library Construction & Sequencing – Amplicons are ligated with adapters and prepared into sequencing libraries, then sequenced using platforms such as Illumina or PacBio.
- Data Analysis – Sequencing data are processed for alignment, variant calling, sequence assembly, or taxonomic classification.
This method is ideal for high-throughput detection of mutations across multiple samples, enabling the simultaneous interrogation of hundreds to thousands of amplicon loci.
Why Use Amplicon Sequencing
Amplicon sequencing is a highly efficient and cost-effective targeted sequencing approach ideal for in-depth analysis of specific genomic regions, microbial communities, or functional gene elements. CD Genomics provides comprehensive, customizable amplicon sequencing solutions—supporting your research from primer design to bioinformatics analysis.
- Highly Targeted & Accurate
By amplifying only the regions of interest with specific primers, this method ensures precise variant detection with high sensitivity—ideal for locus-specific validation and functional studies. - High Throughput & Multiplexing
Capable of analyzing hundreds to thousands of target regions per run, making it suitable for large-scale screening, microbial diversity profiling, and parallel sample processing. - Platform Flexibility & Broad Amplicon Range
Compatible with both Illumina (short-read) and PacBio (long-read) platforms. Supports amplicons from 100 bp up to 10 kb, meeting a wide range of research needs. - Sensitive to Low-Abundance Variants
With ultra-deep sequencing, it excels at detecting low-frequency somatic mutations and resolving complex microbial mixtures, ensuring key insights are not missed.
Amplicon Sequencing vs. Other NGS Methods
Feature | Amplicon Sequencing | Targeted Capture Sequencing | Whole Genome Sequencing (WGS) |
---|---|---|---|
Target Region | Specific PCR-amplified loci | Broader regions via hybrid probes | Entire genome |
Sequencing Depth | Ultra-deep (>1000×) | Moderate to deep (200–800×) | Low to moderate (~30×) |
Data Volume / Cost | Low | Medium | High |
Variant Detection Sensitivity | High (ideal for rare/low-frequency variants) | Medium | Low to medium |
Use Cases | 16S/18S/ITS , CRISPR validation , antibody repertoire | Cancer panels, rare disease genes | Population genetics , structural variants |
Choosing the Right Amplicon Sequencing Platform & Read Length
At CD Genomics, we offer three amplicon sequencing options tailored to your amplicon length, research goals, and data requirements:
Sequencing Type | Amplicon Length | Platform | Read Length | Typical Applications |
---|---|---|---|---|
Standard Amplicon Seq | 100–250 bp | Illumina MiSeq | 2×150 bp | SNP genotyping , editing site validation, rapid strain screening |
Medium-Long Amplicon Seq | 250–550 bp | Illumina MiSeq / NextSeq | 2×250 bp or 2×300 bp | Highly variable regions (e.g., 16S V3-V4), antibody heavy/light chains |
Long Amplicon Seq | >550 bp up to ~10 kb | PacBio Sequel | HiFi CCS (high-fidelity long reads) | Full-length 16S/ITS , paired antibody chains, phasing of variants |
Platform Highlights:
- Illumina Platform: Offers high sequencing accuracy; ideal for short to mid-length amplicons. Supports high multiplexing capacity, making it well-suited for high-throughput studies.
- PacBio Platform: Enables long-read, high-fidelity sequencing. Best suited for structurally complex or long amplicons requiring phasing or full-length analysis.
Recommendations:
- For amplicons <250 bp, we recommend Illumina paired-end 2×150 bp sequencing.
- For amplicons between 250–550 bp, we recommend Illumina 2×250 bp or 2×300 bp configurations.
- For amplicons >550 bp or projects requiring full-length sequences, we recommend the PacBio platform with HiFi (High-Fidelity) reads for single-molecule accurate sequencing.
Our Amplicon Sequencing Process: From Consultation to Reporting
Our modular workflow ensures standardized quality control at every step and allows flexible adjustments based on project needs:
Define targets
Select sequencing platform and depth
Confirm workflow
Sample registration
DNA/PCR quality control
Optional PCR amplification service
Adapter ligation
Indexing and pooling
Library quality check
Illumina or PacBio platforms
Short or long reads
Customizable sequencing depth
Data quality control
Variant detection and taxonomic analysis
Final report delivery
Research Applications of Amplicon Sequencing
Our amplicon sequencing services are widely applied across diverse research fields, enabling precise and efficient analysis of genomic variations and complex sequence information. Key application areas include:
- Genetic Variant Detection
Accurate identification of SNPs, somatic mutations, and complex genetic variants to support various genetic studies and genotyping. - Microbiome Research
Sequencing of 16S rRNA, 18S rRNA, and ITS regions for microbial diversity analysis, phylogenetic profiling, and ecological studies. - Immune Repertoire Sequencing
Targeted sequencing of antibody heavy and light chains to support immune diversity profiling and therapeutic antibody development. - Gene Editing Validation
Assessment of CRISPR/Cas9 editing efficiency and off-target effects to ensure accuracy and reliability in genome editing experiments. - Functional Gene Screening
High-throughput screening of specific gene regions to facilitate gene function studies and novel target discovery. - Plasmid Library Analysis
Comprehensive analysis of plasmid libraries for diversity and structural features to support molecular cloning and genetic engineering.
Amplicon Sequencing Bioinformatics & Data Analysis Services
We offer comprehensive and customizable bioinformatics solutions for amplicon sequencing projects, supporting both short-read and long-read sequencing platforms. Our services help clients unlock the full value of their data and achieve accurate variant detection and functional interpretation.
Short-Read Amplicon Sequencing (Illumina) Analysis
- Data Quality Control & Preprocessing
Removal of adapter sequences and low-quality reads, paired-end read merging to ensure high-quality data. - Denoising & Variant Detection
Advanced algorithms (e.g., DADA2, UNOISE) for noise removal and chimera filtering; accurate identification of SNPs and Indels. - Taxonomic Annotation & Classification
High-efficiency species annotation based on 16S, 18S, ITS, and other curated databases. - Diversity Analysis
Alpha and Beta diversity assessments to support microbial community structure comparison and ecological statistics. - Functional Prediction & Pathway Analysis
Systematic gene function prediction to uncover potential biological functions and metabolic pathways. - Data Visualization & Reporting
Rich graphical outputs and intuitive reports to facilitate in-depth understanding of sequencing results.
Long-Read Amplicon Sequencing (PacBio / ONT) Analysis
- High-Accuracy Long Read Correction
CCS (Circular Consensus Sequencing) and error-correction algorithms enhance read accuracy and data reliability. - Full-Length Amplicon Assembly
Obtain complete amplicon sequences without the need for stitching, enabling accurate detection of complex and long-range variants. - Phasing & Structural Variant Detection
Detect SNPs, Indels, and structural variants from full-length reads, supporting high-resolution variant phasing. - High-Resolution Taxonomic & Functional Annotation
Species and gene function annotation at higher resolution based on full-length sequence alignments. - Advanced Microbial Community Profiling
Leverage full-length reads for in-depth microbial community and functional diversity analysis. - Customized Reporting
Deliverables include structural variation maps, full-length variant details, and comprehensive functional interpretation to support scientific publication.
Sample Requirements and Quality Guidelines for Amplicon Sequencing
To ensure high-quality sequencing results, CD Genomics provides clear guidelines for sample types and input requirements. Below is a quick reference for recommended quantities and quality criteria:
Sample Type | Recommended Input | Purity (OD260/280) | Requirements | Notes |
---|---|---|---|---|
Purified PCR Products | ≥1 µg (min. 500 ng) | 1.8–2.0 | ≥20 ng/μL; high-quality; size matches platform specs | Single, specific band; no non-specific products |
Unpurified PCR Products | Variable | — | Acceptable, but purification is strongly advised for optimal sequencing quality | — |
Fragmented DNA | Sufficient amount | — | Requires uniform size and compatibility with target region | For specific amplicon strategies |
Genomic DNA (gDNA) | ≥500 ng | 1.8–2.0 | High purity; ≥20 ng/μL; no degradation | Ideal for PCR-based amplification |
Restriction Enzyme Cuts | Adequate quantity | — | Complete digestion; free from inhibitors | Suitable for downstream library prep |
Plasmids | Adequate quantity | — | Must be purified; ensure integrity of the target insert | — |
💡 Note: These are general recommendations. For project-specific needs, contact our technical team for tailored guidance.
Why Choose CD Genomics for Amplicon Sequencing?
CD Genomics specializes in delivering high-quality, high-throughput amplicon sequencing services, leveraging advanced Illumina and PacBio platforms to meet diverse amplicon length and complexity requirements. We are committed to providing accurate and reliable variant analysis data, supporting genomic research, microbiome studies, and functional gene screening across various fields.
- Multi-Platform Technology Support
Comprehensive coverage from hundreds to tens of thousands of base pairs using Illumina short reads and PacBio long reads, accommodating diverse experimental designs. - Superior Data Quality and Accuracy
Rigorous quality control processes ensure high coverage and deep sequencing depth for precise detection of low-frequency variants. - Customized Bioinformatics Analysis
Professional sequence assembly, variant detection, and functional annotation with intuitive visualization reports to help clients rapidly extract valuable insights. - End-to-End Professional Support and Fast Response
Experienced team guidance throughout project design, sample QC, sequencing, and data delivery to ensure efficient and smooth project completion.
Partial results are shown below:

The taxonomy distribution of all sample in Phylum classification level.

Species abundance Heatmap.

Rarefaction curve of the sequenced reads for samples (The above figure) & The depth of the sequencing samples (The below figure).

Boxplot analysis based on bray Curtis (A), binary jaccard (B), unweighted unifrac (C), and weighted unifrac (D).

PCoA analysis based on bray Curtis (A), binary jaccard (B), unweighted unifrac (C), and weighted unifrac (D).

UPGMA clustering tree.

Mean proportion of treated and control group.

Cladogram.

LDA SCORE.
1. What is the difference between targeted sequencing and amplicon sequencing?
Amplicon sequencing involves the PCR amplification of specific genomic regions followed by sequencing, which ensures high specificity and on-target rates due to the precise design of primers. It is particularly suitable for analyzing small, defined regions of the genome, such as in genetic variation analysis and microbial profiling. In contrast, targeted sequencing encompasses methods like hybrid capture and probe-based enrichment to selectively sequence larger genomic regions or multiple genes without prior amplification. This allows for a more comprehensive analysis of selected areas, but may have variable on-target rates depending on the efficiency of the enrichment process. Amplicon sequencing, by its nature, achieves superior on-target rates in contrast to other targeted sequencing methodologies, attributing this efficiency to the precise design of primers. This approach finds particular applicability in tasks like genotyping via sequencing, as well as the discernment of germline single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and known genetic fusions.
2. What are the primary applications of Amplicon Sequencing?
Amplicon sequencing serves as a pivotal tool in diverse scientific domains, encompassing but not limited to the following applications:
- Genetic Variance Analysis: Unveiling single nucleotide polymorphisms (SNPs), insertions, deletions, and other hereditary genetic modifications.
- Microbiome Investigations: Profiling microbial communities by sequencing marker genes like the 16S ribosomal RNA.
- Oncological Inquiry: Spotting somatic mutations and genetic modifications within tumor specimens.
- Hereditary Disease Research: Exploring the genetic underpinnings of inherited disorders.
- Environmental Surveys: Gauging biodiversity and identifying specific organisms within environmental specimens.
3. What is the difference between Amplicon Sequencing and Whole-Genome Sequencing (WGS)?
Amplicon sequencing targets specific genomic regions by amplifying them with PCR before sequencing, allowing for high specificity and depth in analyzing small, defined regions, such as in detecting mutations or profiling microbial communities. In contrast, whole-genome sequencing (WGS) sequences the entire genome without prior selection or amplification, providing a comprehensive view of all genetic information, which is ideal for discovering novel variants and obtaining a complete genetic profile, but it is more resource-intensive and less focused on specific areas of interest.
4. How do you choose the target regions for Amplicon Sequencing?
The selection of target segments in Amplicon Sequencing hinges on the study's objectives and the biological significance of these segments. Pertinent factors include associations with diseases, genetic markers, regions of notable variability, and functional relevance. Collaborating with bioinformaticians and utilizing databases like dbSNP and ClinVar can facilitate precise target region identification.
5. What types of bioinformatic analyses can be performed with Amplicon Sequencing data?
- Variant identification: Recognizing SNPs, insertions, deletions, and diverse genetic variances.
- Analysis of microbial diversity: Evaluating the constitution and prevalence of microbial consortia.
- Phylogenetic investigation: Researching evolutionary connections among sequences.
- Functional elucidation: Associating genetic variances with plausible functional implications.
6. Can Amplicon Sequencing detect rare variants?
Without a doubt, Amplicon Sequencing displays notable sensitivity, allowing the detection of infrequent variants found at low occurrences. This trait renders it applicable for situations like the spotting of mutations in cancer and the evaluation of microbial diversity.
7. How does CD Genomics ensure sequencing success with high-GC content regions?
We use a 3-layer strategy to maximise yield and accuracy:
- Optimised Library Prep:
Methylation-adaptive polymerases reduce GC bias during amplification. - Enhanced QC Monitoring:
Real-time nanopore sensing ensures intact, high-quality fragments. - Bioinformatics Correction:
Advanced algorithms compensate for GC-skewed abundance in downstream data.
Customer Publication Highlight
Microbial adaptation and response to high ammonia concentrations and precipitates during anaerobic digestion under psychrophilic and mesophilic conditions
Journal: Water Research
Published: 1 October 2021
DOI: https://doi.org/10.1016/j.watres.2021.117596
Background
High ammonia concentrations (TAN >1.5 g/L) are a major cause of methane inhibition in anaerobic digestion (AD), particularly under mesophilic (37°C) and psychrophilic (22.6°C) conditions. Phosphate precipitates (e.g., struvite) further exacerbate system collapse, reducing methane yield by >50%. This study pioneers the exploration of microbial ammonia adaptation mechanisms in psychrophilic reactors and analyzes the long-term impact of precipitates on methanogenic communities.
Project Objectives
- Microbial Adaptation: Uncover microbial responses to high TAN (4,000 mg/L) in psychrophilic vs. mesophilic AD.
- Key Consortium Identification: Identify ammonia-tolerant methanogens and precipitate-sensitive taxa.
- Functional Dynamics: Link metagenomic shifts to methane metabolism pathways.
CD Genomics’ Services
As the core genomics partner, CD Genomics delivered:
- 16S rRNA Amplicon Sequencing
Platform: Illumina MiSeq PE300
Target Region: V4-V5 hypervariable (optimized for Archaea detection).
Primers: 515F (5′-GTGYCAGCMGCCGCGGTAA) / 926R (5′-CCGYCAATTYMTTTRAGTTT).
Data Depth: ~30,000 reads/sample (covering all TAN adaptation phases). - Whole-Genome Metagenomic Sequencing
Platform: Illumina NovaSeq PE150.
Depth: 6 GB raw data/sample (initial vs. endpoint samples).
DNA Standards: Concentration ≥50 ng/μL, 260/280 ratio <1.8. - Bioinformatics Analysis
Pipeline: MG-RAST v4.0.3.
Taxonomy: SILVA (16S classification), GenBank (metagenome classification).
Functional Annotation: KEGG/Subsystems databases (e-value ≤10⁻²⁵, 90% identity).
Diversity Metrics: Shannon Index, PCoA (Bray-Curtis distance).
Key Findings
- Ammonia Adaptation in Psychrophilic Reactor
- Dominant Methanogen Shift:
- At TAN >3 g/L, Methanocorpusculum reached 71% relative abundance (metagenome data), becoming the dominant hydrogenotrophic methanogen (Fig. 5a).
- Bacterial consortium shifted to Enterococcaceae (Firmicutes), surging from 0.1% to 80% abundance (Fig. 4a).
- Functional Resilience:
- KEGG analysis revealed enhanced methane metabolism genes (e.g., methyl-compound conversion) under high TAN (Fig. 8).
- Dominant Methanogen Shift:
- Precipitate-Induced Collapse in Mesophilic Reactors
- Methane Yield: Declined by >50% post-precipitate formation with no recovery within 50 days (p<0.05).
- Methanogen Depletion:
- Annotated methanogen genes collapsed from >300,000 to <2,500 hits (metagenome data, Fig. 5b).
- Species Replacement: Methanosarcina barkeri displaced M. mazei as the dominant ammonia-tolerant archaeon (Fig. 4b).
- α Diversity as Stability Indicator
- Psychrophilic Reactor: Diversity decreased by 40% (Shannon Index) upon successful ammonia adaptation (Fig. 3a).
- Mesophilic Reactor: Stable diversity masked functional failure, evidenced by persistent methane decline (Fig. 3b).
Figures Referenced
Figure 3. Alpha diversity and methane yield in experimental reactors at different ammonia concentrations (a) psychrophilic reactor (R1-CO) and (b) mesophilic reactor (R2-WW).
Figure 4. Bacteria and Archaea profiles from 16S rRNA amplicon data along with the step-by-step increase in ammonia levels.
Figure 5. (a) Relative abundance of Archaea Domain, and (b) number of hits for Bacteria and Archaea Domain in psychrophilic (R1-CO) and mesophilic (R2-WW) AD.
Figure 8. Number of genes copy for methane metabolism (anabolism and catabolism) using KEGG Database.
Implications
- Process Optimization: Enriching Methanocorpusculum in psychrophilic AD enhances ammonia tolerance, reducing heating energy demands.
- Risk Mitigation: Struvite precipitation causes irreversible methanogenic inhibition, necessitating real-time phosphate/pH monitoring.
- Biotechnological Resource: M. barkeri and Methanocorpusculum serve as candidates for ammonia-resistant inoculum development.
Here are some publications that have been successfully published using our services or other related services:
Distinct functions of wild-type and R273H mutant Δ133p53α differentially regulate glioblastoma aggressiveness and therapy-induced senescence
Journal: Cell Death & Disease
Year: 2024
High-Density Mapping and Candidate Gene Analysis of Pl18 and Pl20 in Sunflower by Whole-Genome Resequencing
Journal: International Journal of Molecular Sciences
Year: 2020
Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI
Journal: New phytologist
Year: 2017
Generation of a highly attenuated strain of Pseudomonas aeruginosa for commercial production of alginate
Journal: Microbial Biotechnology
Year: 2019
Combinations of Bacteriophage Are Efficacious against Multidrug-Resistant Pseudomonas aeruginosa and Enhance Sensitivity to Carbapenem Antibiotics
Journal: Viruses
Year: 2024
Genome Analysis and Replication Studies of the African Green Monkey Simian Foamy Virus Serotype 3 Strain FV2014
Journal: Viruses
Year: 2020
See more articles published by our clients.