Amplicon Sequencing Services

Amplicon sequencing is based on NGS technology. The ultra-deep sequencing of amplicons (PCR products) allows efficient variant identification and characterization.

This technique has a wide range of applications, including 16S/18S/ITS gene sequencing, SNP genotyping, genome editing clone verification, somatic/complex variant discovery, and antibody heavy or light chain et al..

Whether you would like to detect the diversity of microbial communities or discover rare somatic mutations in complex samples. CD Genomics could provide professional, cost-efficient and high-speed amplicon sequencing services to meet your project requirements.

Key Features and Advantages

Providing high-sensitive detection levels through ultra-deep sequencing
Achieving high coverage by sequencing hundreds of thousands of amplicons per reaction
Cost-efficient and fast turnaround time
Microbial culture is not necessary
Wide range of amplicons from 100 bp to 10 kb

Sample Types

Purified or unpurified PCR products, fragmented DNA, gDNA, restriction digested material, and plasmids.

Sequencing Workflow

SNP Microarray

Fig.1 Sequencing workflow.

Sequencing strategies CD genomics recommended

Table 1. Strategies for amplicon sequencing.

Amplicon Size	Applications	Sequencing Configuration
100-250 bp	SNP Genotyping Genome editing clone verification Somatic/complex variant discovery	Illumina PE 150
250-550 bp	Antibody heavy or light chain 16S/18S/ITS gene sequencing	Illumina PE 250/300
>550 bp	Paired antibody heavy and light chains Full-length 16S Variant phasing	PacBio Sequel

Information analysis process

For diversity detection

Data quality control
OTU clustering
Alpha Diversity (Sample complexity analysis)
Beta Diversity (Multi-sample comparative analysis, PCoA/PCA/NMDS/Simper)
Analysis of species differences between groups (T-test/Metastat/LEfSe)
Analysis of community structure differences between groups (Anosim/MRPP/Adonis/Amova)
Correlation analysis of environmental factors (Spearman/Mantel test/CCA/RDA/dbRDA/VPA)
Network analysis
Function prediction (PICRUSt/FAPROTAX/Tax4fun/FunGuild)
Advanced analysis (enterotype analysis/ Bayesian analysis)

For variant identification

SNP Microarray

Fig.2 Data analysis for variant identification.

References

Betge J, Kerr G, Miersch T, et al. 2015. Amplicon sequencing of colorectal cancer: variant calling in frozen and formalin-fixed samples. Plos One 10:e0127146.
Ison SA, Delannoy S, Bugarel M, et al. 2016. Targeted amplicon sequencing for single-nucleotide-polymorphism genotyping of attaching and effacing Escherichia coli O26:H11 cattle strains via a high-throughput library preparation technique. Appl Environ Microbiol 82:640-649.