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

1. Target Selection & Primer Design – Primers are designed to amplify specific genomic regions of interest based on research objectives.
2. PCR Amplification – The target regions are amplified from sample DNA to generate high-abundance amplicons.
3. Library Construction & Sequencing – Amplicons are ligated with adapters and prepared into sequencing libraries, then sequenced using platforms such as Illumina or PacBio.
4. 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. N2 Jenomics Lab Pvt. Ltd. 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

Choosing the Right Amplicon Sequencing Platform & Read Length

At N2 Jenomics Lab Pvt. Ltd. , we offer three amplicon sequencing options tailored to your amplicon length, research goals, and data requirements:

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:

Project Consultation

Define targets

Select sequencing platform and depth

Confirm workflow

Sample Submission & QC

Sample registration

DNA/PCR quality control

Optional PCR amplification service

Library Preparation

Adapter ligation

Indexing and pooling

Library quality check

Sequencing

Illumina or PacBio platforms

Short or long reads

Customizable sequencing depth

Bioinformatics & Reporting

Data quality control

Variant detection and taxonomic analysis

Final report delivery

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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, N2 Jenomics Lab Pvt. Ltd. provides clear guidelines for sample types and input requirements. Below is a quick reference for recommended quantities and quality criteria:

💡 Note: These are general recommendations. For project-specific needs, contact our technical team for tailored guidance.

Why Choose N2 Jenomics Lab Pvt. Ltd. for Amplicon Sequencing?

N2 Jenomics Lab Pvt. Ltd. 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.

Demo Results

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.

Amplicon Seq FAQs

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 N2 Jenomics Lab Pvt. Ltd. ensure sequencing success with high-GC content regions?

We use a 3-layer strategy to maximise yield and accuracy:

1. Optimised Library Prep:
   Methylation-adaptive polymerases reduce GC bias during amplification.
2. Enhanced QC Monitoring:
   Real-time nanopore sensing ensures intact, high-quality fragments.
3. Bioinformatics Correction:
   Advanced algorithms compensate for GC-skewed abundance in downstream data.

Amplicon Seq Case Studies

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

1. Microbial Adaptation: Uncover microbial responses to high TAN (4,000 mg/L) in psychrophilic vs. mesophilic AD.
2. Key Consortium Identification: Identify ammonia-tolerant methanogens and precipitate-sensitive taxa.
3. Functional Dynamics: Link metagenomic shifts to methane metabolism pathways.

N2 Jenomics Lab Pvt. Ltd. ’ Services

As the core genomics partner, N2 Jenomics Lab Pvt. Ltd. delivered:

1. 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).
2. 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.
3. 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

1. 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).
2. 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).
3. 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

1. Process Optimization: Enriching Methanocorpusculum in psychrophilic AD enhances ammonia tolerance, reducing heating energy demands.
2. Risk Mitigation: Struvite precipitation causes irreversible methanogenic inhibition, necessitating real-time phosphate/pH monitoring.
3. Biotechnological Resource: M. barkeri and Methanocorpusculum serve as candidates for ammonia-resistant inoculum development.