Amplicon Sequencing Services

What is Amplicon Sequencing

Amplicon sequencing involves sequencing specific lengths of PCR products or captured fragments to analyze variations within the sequences. This technique allows for high-coverage sequencing of target regions tailored to different requirements and can detect low-frequency mutations. Currently, amplicon sequencing primarily encompasses functional gene sequencing and targeted sequence-directed capture fragment sequencing.

Amplicon sequencing is based on NGS technology or PacBio SMRT sequencing. 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, CRISPR sequencing, somatic/complex variant discovery, antibody screening sequencing, immune repertoire sequencing, 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.

Advantages of Amplicon Sequencing

• 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

Amplicon Sequencing Workflow

CD Genomics employs multiple platforms to provide the fast and accurate amplicon sequencing services and bioinformatics analysis. Our highly experienced experts execute quality management, following every procedure to ensure high quality results. The general workflow for amplicon sequencing is outlined below.

Service Specifications

	Sample Requirements Purified or unpurified PCR products Purified Amplicon≥ 1 µg, Minimum Quantity: 500 ng, Concentration≥ 20 ng/µL Fragmented DNA gDNA Restriction digested material Plasmids Note: Sample amounts are listed for reference only. For detailed information, please contact us with your customized requests.
Click	Sequencing Amplicon Size: 100-250 bp: SNP Genotyping, Genome editing clone verification, Somatic/complex variant discovery; Illumina PE 150 Amplicon Size: 250-550 bp: Antibody heavy or light chain, 16S/18S/ITS gene sequencing; Illumina PE 250/300 Amplicon Size >550 bp: Paired antibody heavy and light chains, Full-length 16S, Variant phasing; PacBio Sequel
	Bioinformatics Analysis We provide customized bioinformatics analysis including: 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) Note: Recommended data outputs and analysis contents displayed are for reference only. For detailed information, please contact us with your customized requests.

Analysis Pipeline

Amplicon sequencing facilitates various bioinformatic analyses. Depending on your specific needs, we can conduct the necessary data analyses. For instance, the data analysis pipeline for amplicon sequencing can be exemplified by variant identification.

Deliverables

• The original sequencing data
• Experimental results
• Data analysis report
• Details in Amplicon Sequencing for your writing (customization)

References:

1. 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.
2. 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.

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.

Full-length 16S rRNA gene sequencing reveals spatiotemporal dynamics of bacterial community in a heavily polluted estuary, China

Journal: Environmental Pollution

Impact factor: 8.071

Published: 15 April 2021

Background

The Liaohe Estuary faces severe inorganic nitrogen and heavy metal pollution, impacting its ecosystem and marine biodiversity. This study uses SMRT sequencing to analyze bacterial community dynamics across seasons and lifestyles, aiming to understand the impact of environmental factors and pollution on the estuarine ecosystem.

Methods

Results

After filtering, 449,089 clean reads were generated from sediment and seawater samples, with sediment and seawater yielding 9,803 and 17,950 OTUs respectively. Alpha diversity was higher in sediment samples than seawater samples in both wet and dry seasons. Bacterial communities differed between sediment and seawater, with distinct dominant phyla in each. Seasonal and habitat variations significantly influenced microbial community structures, with lifestyle-based differences observed mainly in the dry season.

Clustering and PCoA analyses revealed significant spatial differences in sediment bacterial communities between the N and O areas, regardless of season, with Gillisia being the core bacterium in the N area.

In seawater, bacterial community relationships were more complex due to space, season, and lifestyle factors, with significant differences observed among free-living (FL) and particle-attached (PA) bacteria, especially in the O area and dry season. Specific bacteria showed seasonal lifestyle transitions, and environmental factors like Cu, Zn, and salinity significantly influenced community structures.

Fig 1. CCA of the relationship between environmental factors and the bacterial community at the species level. Heatmap of Spearman's rank correlation between bacterial genus and chemical characteristics in sediment (D) and seawater (E) samples.

Conclusion

This study reveals distinct bacterial communities in sediment and seawater of the Liaohe Estuary, influenced by habitat, space, season, and lifestyle, with heavy pollution significantly impacting nearshore bacterial diversity and composition.

Reference:

1. Hongxia M, Jingfeng F, Jiwen L, et al. Full-length 16S rRNA gene sequencing reveals spatiotemporal dynamics of bacterial community in a heavily polluted estuary, China. Environmental Pollution, 2021, 275: 116567.