Amplicon Sequencing Service

Traditional Sanger sequencing lacks the throughput and sensitivity for analyzing multiple targets or detecting rare variants in complex samples. Amplicon sequencing overcomes this by combining targeted PCR amplification with next-generation sequencing, enabling high-sensitivity, parallel analysis of specific genomic regions with deep coverage. CD BioSciences provides comprehensive amplicon sequencing services, supporting research in oncology, microbiology, genetics, and functional genomics with tailored solutions.

Introduction to Amplicon Sequencing

Amplicon sequencing is a highly focused next-generation sequencing (NGS) approach designed for in-depth analysis of user-defined genomic regions. The core methodology involves designing sequence-specific primers to amplify target DNA or cDNA regions through polymerase chain reaction, followed by high-throughput sequencing of these amplified fragments. This targeted strategy achieves exceptionally deep coverage at the loci of interest, making it uniquely powerful for detecting low-frequency genetic variants, profiling microbial communities via marker genes (e.g., 16S/ITS), and quantifying post-transcriptional RNA modifications such as adenosine-to-inosine (A-to-I) editing. It serves as a cost-effective and rapid solution for applications requiring precision and sensitivity beyond broader sequencing methods.

Fig.1 Demonstration of the workflow for the 16S rRNA and ITS amplicon sequencing. (Athanasopoulou K, et al., 2023)

Features of Amplicon Sequencing

Amplicon sequencing is engineered for focused genetic interrogation, delivering unparalleled depth and precision at user-specified genomic loci. By concentrating sequencing power on defined targets, this method transforms complex biological questions into manageable, high-resolution data sets. Its design flexibility and analytical sensitivity make it the preferred platform for applications where accuracy, cost-efficiency, and throughput are paramount, including rare variant detection, microbial community profiling, and quantitative RNA modification analysis.

1. High Sensitivity for Rare Variants: Capable of detecting mutations or rare alleles present at frequencies as low as 1% in heterogeneous samples.
2. Cost-Effective Targeted Analysis: Maximizes data yield and value by concentrating sequencing resources on specific regions of interest.
3. Flexible Multiplexing: Supports simultaneous analysis of hundreds of target regions or samples in a single run, enhancing throughput.
4. Precise Quantification: Enables accurate measurement of allele frequencies, microbial abundances, or RNA editing ratios at single-nucleotide resolution.

Our Services

CD BioSciences offers tailored amplicon sequencing services, from custom panel design to advanced data interpretation. Our optimized workflows ensure high specificity and reproducibility for your project, whether focused on somatic variant discovery in cancer, 16S rRNA-based microbiome characterization, or quantitative analysis of RNA adenosine-to-inosine (A-to-I) editing events. We provide the depth and accuracy needed to uncover critical biological insights from diverse sample types.

Workflow of Amplicon Sequencing

Our amplicon sequencing service follows an integrated, end-to-end pipeline optimized for accuracy, sensitivity, and scalability. From initial project design to final data interpretation, each step is meticulously controlled to ensure reliable and reproducible results tailored to your specific research objectives, whether targeting genetic variants, microbiome composition, or RNA modifications.

Project Consultation & Panel Design

Collaborative design of target-specific primers or selection from optimized curated panels for genes, microbial marker regions (e.g., 16S, ITS), or RNA editing sites (e.g., A-to-I loci).

Nucleic Acid Extraction & QC

High-quality DNA or RNA extraction using validated protocols, followed by rigorous spectrophotometric and fluorometric quantification, and integrity assessment (e.g., Bioanalyzer, TapeStation) to ensure input suitability.

Target Amplification & Library Preparation

Multiplex PCR amplification of target regions using high-fidelity polymerases, followed by enzymatic cleanup, adapter ligation, and incorporation of dual-index barcodes for efficient sample pooling and multiplexed sequencing.

High-Throughput Sequencing & QC

Pooled libraries are sequenced on Illumina platforms (NovaSeq, MiSeq, or NextSeq) with read lengths and depth optimized for the application, generating high-quality raw data with embedded sample identifiers.

Bioinformatic Analysis & Reporting

Data is processed through specialized pipelines including demultiplexing, primer trimming, read alignment, and application-specific analysis (variant calling, OTU/ASV clustering, or editing efficiency quantification), culminating in a comprehensive report with annotated results and visualizations.

Supported Sample Types

1. Human & Animal Biospecimens: Blood, saliva, tissue biopsies (fresh/frozen/FFPE), cell lines, circulating cell-free DNA (cfDNA), formalin-fixed paraffin-embedded (FFPE) blocks.
2. Microbial & Environmental Samples: Bacterial cultures, stool, soil, water, air filters, swabs, biofilm samples.
3. Plant & Agricultural Samples: Leaf, seed, root, and stem tissues; gDNA from crops or model plants.
4. RNA Samples for Editing Analysis: High-quality total RNA or poly(A)-selected RNA from any biological source for targeted cDNA analysis.
5. Low-Input & Challenging Samples: Single cells, laser-capture microdissected (LCM) samples, forensic traces, ancient DNA (with specialized extraction protocols).

Our Advantages

1. Application-Optimized Design: Expertise in designing panels for diverse goals, from cancer gene hotspots to conserved RNA editing loci.
2. Ultra-Sensitive Detection: Optimized wet-lab and bioinformatic protocols to minimize noise and maximize signal for low-frequency events.
3. End-to-End Project Support: Dedicated support from initial design through final data interpretation and biological contextualization.
4. Rigorous Quality Control: Implementation of controls at each step to ensure data accuracy and reliability, critical for clinical and translational research.

Amplicon sequencing is an ideal technique for high-throughput, highly sensitive quantitative analysis of specific RNA sites, such as A-to-I editing loci. Furthermore, our LC-MS/MS platform offers an orthogonal analytical dimension, enabling global identification and absolute quantification of modified nucleosides, including inosine resulting from A-to-I editing, thereby providing validation and a complementary perspective to sequencing data for more comprehensive and reliable integrated epitranscriptomic analysis. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

Reference

1. Athanasopoulou K, Adamopoulos P G, Scorilas A. Unveiling the human gastrointestinal tract microbiome: the past, present, and future of metagenomics[J]. Biomedicines, 2023, 11(3): 827.