- Sequence Smarter / Amplicon Sequencing
Amplicon Sequencing
Understand which microbes are present and
their relative abundance.
High-throughput amplicon sequencing services leverage Illumina and Nanopore sequencing platforms to deliver accurate taxonomic identification and relative abundance measurements across a wide range of sample types.
Amplicon sequencing
Amplicon sequencing provides a rapid and cost-effective way to understand community composition before advancing to more comprehensive shotgun metagenomic sequencing projects that can gather even more information about mixed-species populations, including functional annotation, strain characterization, antimicrobial resistance genes, and metabolic potential.
Why use amplicon sequencing at SeqCoast?
Cost-effective method for profiling complex microbial communities
Scalable throughput, from dozens of samples to tens of thousands
Availability of custom primers, PNA clamps, etc. for specialized applications
Illumina Amplicon Sequencing
- High-accuracy 2 x 300 bp reads leveraging X-LEAP sequencing chemistry
- Overlapping paired reads maximize assignment accuracy
- Ideal for community diversity studies and relative abundance profiling
Nanopore Full-length Amplicon Sequencing
- Full-length Nanopore reads (1,500 bp+)
- Compatible with custom primer designs, including ITS and other amplicons
- Ideal when genus-level identification is insufficient
Our standard primer sets are carefully selected to maximize taxonomic coverage across diverse microbial communities, enabling robust and consistent detection of organisms from a wide range of sample types.
Applications
Example sequencing targets:
- Human-associated microbiomes (gut, skin, oral, vaginal)
- Soil and rhizosphere
- Marine microbial communities
- Wastewater
- Fermentation systems
- Synthetic microbial consortia
- eDNA sampling
Example applications where amplicons excel
- Microbiome characterization
- Community composition analysis
- Environmental monitoring
- Food and beverage surveillance
- Biodiversity assessments
- Gut microbiome perturbation studies
- Low-barrier pilot sequencing before launching full metagenomics project
Does your project require non-standard primers? Get in touch!
SeqCoast features and benefits
Our expertise and high-throughput capabilities make us the ideal partner for custom projects of any size or complexity, whether you have dozens or thousands of samples.
Flexibility at scale
- High-throughput sequencing, with capacity to process hundreds of thousands of samples.
- Data volumes ranging from 10,000 reads to 100,000+ reads per sample.
Gold-standard library preparation and sequencing
- Industry-leading sequencing chemistries and reagents – no off-brands, homemade enzymes, or compromised quality.
- Established and validated 16S and ITS workflows.
- Custom primer workflows overseen by PhD-level microbiologists.
Analysis options
- Complimentary basic relative abundance analysis for every amplicon sequencing order.
- Upgrade to our advanced amplicon analysis for group comparisons, host sequencing depletion, and more!
Multi-point QC
- Samples inspected upon arrival to ensure they have arrived intact and conform to biosafety standards.
- Sequencing run quality metrics are evaluated to ensure high-quality results.
- Custom QC services available upon request for large orders.
Need custom analysis?
Most projects are covered by our standard analysis pipelines. If your project requires custom analysis, tell us about your project goals and needs!
How to prep samples for submission
Send raw samples
- Send raw samples for our robust DNA extraction workflow.
Or send purified DNA
- Send >10ng/ul of DNA in a volume of >20ul eluted in nuclease-free water or dilute TE.
- We accept samples that have lower concentrations/volumes, but they may have a lower chance of success.
Quantification and elution
- We strongly recommend quantifying by Qubit or other fluorometric method, as other methods (such as Nanodrop or Tapestation) are often wildly inaccurate.
- We recommend eluting samples in nuclease-free water or diluted TE, but we do accept other elution buffers.
- Excessive EDTA and extraction contaminants (salts, phenol, polysaccharides, etc.) can cause sample failure.
48+ samples
- We offer our COMPASS system for CoOrdinated Management and Processing for Accelerated Sequencing Services.
- COMPASS allows for the fastest processing and the lowest price!
- You will receive a custom shipping container with validated plasticware, stabilization buffer, & prepaid shipping label at no additional charge.
24-48 samples
- Must be sent in a 96-well plate or individually-capped 8-tube strips.
- 96-well plates should be fully-skirted and sealed well with a foil seal (plastic seals are very prone to leaking during shipping).
<24 samples
- Individually-capped 8-tube strips (preferred) or 1.5 mL tubes.
96-well plates
- Label with Order Number and (if necessary) Plate Number.
Tubes
- Label caps with SeqCoast Sample ID (assigned in your Sample Manifest).
- Labeling tubes with your own sample names instead of the SeqCoast Sample IDs can cause delays!
Please send
All samples must be securely packaged in a clean, organized fashion using secondary containment. Leaking samples will be immediately disposed of, as required by our biosafety measures.
Please do not send
Bags of tubes, individual tubes wrapped in parafilm, large-format tubes (e.g. 50ml conicals), non-sterile/soft-sided containers (e.g. plastic bags), unsealed cell culture 96- well plates. We’ve seen it all! Please double-check your shipments to ensure your samples make it to us safe and sound.
Data delivery, security, and access
We make it easy to access your data!
Most Illumina orders are completed within 10 business days of sample receipt.
Data delivery options include SFTP Download, AWS transfer, Box.com, and alternatives on request.
Data are stored on AWS cloud infrastructure in the United States, providing scalable storage, high availability, and industry-recognized security controls.
Pricing — the more you order, the lower the price!
How much data do I need for amplicon sequencing?
If you are unsure about the data volume per sample you need, we recommend that you do one of the following:
- Perform a literature search for similar sample types and analyses.
- Send pilot samples aiming for more data than you think you need, then use down-sampling methods to determine the optimal data volume for your full experiment.
While we do not provide specific recommendations for every project, please see the following table for illustrative examples of data volume tiers that our customers have used:
- Illumina 16S V3/V4 20k reads, 2x300bp
- Nanopore 16S full-length 10k reads
- Illumina 16S V3/V4 100k reads, 2x300bp
- Nanopore 16S full-length 50k reads
Frequently Asked Questions
How should I extract my DNA?
Let us do the hard work for you! We offer DNA extraction using bead beating lysis.
If you want to tackle it yourself, many column-based extraction kits provide good results, as do other methods if care is taken to remove contaminants. Our library prep methods work best with buffers containing less than 1mM EDTA.
Can I send a lower concentration or volume than your recommendations?
Yes, we are often successful with lower-concentration samples and are happy to process any samples that you send!
Please note that samples that do not meet our recommendations have a lower chance of success.
Custom low-input library preparation services are available for large orders.
How much sequencing data do I need?
See the “How Much Sequencing Data Do I Need for Amplicon Sequencing?” section above.
How do you manage multiplexing? Do I have to worry about index hopping?
We use industry best practices, because we know that accurate sample identification and read assignment are critical for microbial community studies.
For Illumina amplicon sequencing, we exclusively use unique dual indexes (UDIs). Unlike combinatorial indexing approaches, UDIs effectively eliminate index misassignment due to index hopping, ensuring that reads are assigned to the correct sample with the highest possible confidence.
For Nanopore amplicon sequencing, we require matching barcode assignments on both ends of each read. This dual-barcode validation approach effectively eliminates barcode misassignment and maintains accurate sample demultiplexing. Not all providers do this!
These strategies allow us to effectively scale even very large projects with confidence.
Do you perform sequencing for custom amplicons (e.g. CO1, 18S, 16S V4 only)?
Yes! We offer custom amplicon sequencing for large orders. Contact Us
My sample has a lot of host DNA present. Is there anything I can do to prevent off-target amplification of mitochondria and chloroplasts?
Yes! We offer peptide nucleid acid (PNA) clamps for large orders. Contact Us
PNA clamps bind to specific DNA target regions and block amplification of that region. PNA clamps can be used to dramatically reduce off-target amplification, especially in samples with a large proportion of unwanted host DNA.
Do you have requirements for A260/230 and A260/280 absorbance ratios?
Our library preparation workflows are generally quite robust and can tolerate moderate deviations from ideal absorbance ratios.
We do not have requirements surrounding absorbance ratios and will not provide recommendations on whether your ratios are sufficient.
However, please be aware that lower-quality samples have lower chances of success and may require that you perform a cleanup prior to sending them for sequencing.