- Sequence Smarter / Hybrid Illumina and Nanopore Sequencing
Hybrid Illumina and Nanopore
Sequencing for complete bacterial genomes
Hybrid sequencing combines the power of Illumina and Nanopore sequencing, ideal for high-quality genome assembly, annotation, and variant calling. Sequencing at batch sizes from dozens of samples to tens of thousands.
Hybrid Sequencing
SeqCoast provides high-throughput hybrid whole-genome sequencing (Illumina short reads and Nanopore long reads) for microbial isolates, mixed populations, and metagenomes. We run NextSeq 2000/NovaSeq X platforms for Illumina sequencing and the PromethION platform for Nanopore sequencing.
Hybrid sequencing leverages the quality of short reads and the read lengths of long reads to allow for robust, closed genome assemblies. Our standard Illumina whole-genome sequencing services provide 2×150 bp paired reads, using modern X-LEAP chemistry for high read accuracy and high output per run.
Our Nanopore approach sequences reads up to 100 kbp and occasionally much longer, limited mainly by the length of the DNA you send. We use Nanopore’s most recent R10 sequencing chemistry for high read accuracy.
Why choose hybrid sequencing at SeqCoast?
Our standard Illumina whole-genome sequencing provides 2×150 bp paired-end reads with high read accuracy.
Nanopore reads can reach 100 kbp or longer, depending primarily on the length of the DNA you provide.
We use Illumina X-LEAP and Nanopore R10 chemistry to generate accurate sequencing data.
Combining short-read accuracy with long-read coverage helps resolve complex regions and supports closed genome assemblies.
Applications
Example microbial sequencing targets:
- Microbial isolates: Bacteria, Fungi (yeast, filamentous), Protozoans, Single-cell eukaryotes (e.g. algae, amoeba)
- DNA phage or other viruses
- Whole populations (e.g. experimental evolution, intrastrain diversity)
- Metagenomes (e.g. microbiome, soil, contrived communities, wastewater)
Example applications for hybrid sequencing
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Assembly and annotation
- Long reads can span difficult-to-assemble regions, supporting robust, single-contig genome assemblies.
- High quality short reads improve the confidence in each base call.
- Together, these two strategies produce more complete and accurate assemblies than either alone.
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Reference-based variant calling for clones or populations
- Short-read variant calling is our standard offering and is a long-standing technique that excels at identifying SNPs.
- Long reads can be used to identify large insertions, deletions, inversions, and translocations - Inquire about this custom offering!
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 200 Mbp to 50 Gbp+ per sample.
Gold-standard library preparation and sequencing
- Illumina library prep and sequencing reagents. No off-brands, homemade reagents, or compromised quality.
- Robust, tagmentation-based library preparation that can effectively handle a wide range of GC contents. We exclusively use unique dual indexes (UDIs) to prevent index hopping.
- Ligation-based Nanopore library preparation to preserve the length of your DNA and bypass PCR-related errors. Not all providers use this method!
- High throughput and high read accuracy. Q30+ for Illumina (99.9%+ accuracy) and Q20+ for Nanopore (99%+ read accuracy).
Multi-point QC
- All samples checked upon arrival to ensure they have arrived intact and conform to biosafety standards.
- DNA fragment length distributions and library concentrations are measured to ensure appropriate library pool balancing and optimize data output for each sample.
- Sequencing run quality metrics checked by a qualified expert.
- Custom QC services available upon request for large orders.
Need custom sequencing?
Most projects are covered by our standard analysis pipelines above. If your project requires custom analysis, tell us about your project goals and needs!
How to prep samples for submission
Our hybrid bundles include our DNA extraction service at no extra cost!
- Read more about our robust DNA extraction workflow.
Or alternatively
- Send >50 ng/ul of DNA in a volume of >50 ul 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 hybrid sequencing orders are completed within 14 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 sequencing data do I need for hybrid 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:
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.
Our DNA extraction service can isolate high-molecular-weight DNA from a wide range of sample types and can make all the difference for getting high-quality sequencing results!
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.
Remember that you want long pieces of DNA to make the most of your long-read sequencing.
While harsh methods like bead beating are often necessary for tough-to-lyse microbes, we recommend that you take any reasonable steps to reduce the shearing of your DNA during this process.
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.
How long should my DNA be for hybrid sequencing?
Nanopore sequencing can handle any length of DNA, but most applications benefit from providing DNA that is as long as possible. Specific requirements will depend on your particular sample type and project goals, but most customers aim to send DNA with average fragment lengths of ~10,000 bp or longer.
How much sequencing data should I order?
See the “How Much Sequencing Data Do I Need for Hybrid Sequencing?” section above.
Is the Illumina library preparation tagmentation-based or ligation-based?
Our standard Illumina library preparation is tagmentation-based and uses gold-standard reagents from Illumina.
Does the Nanopore library preparation include PCR amplification or shearing?
No, our Nanopore library preparation does not include amplification or shearing. It is ligation-based, so your original DNA strands are what gets sequenced.
Do you perform sequencing for phage and other viruses?
Our standard services are usually compatible with dsDNA phages and other dsDNA viruses!
Please note that phages sometimes have highly-modified DNA that can cause issues during Nanopore sequencing. In these cases, Illumina sequencing has a higher chance of success.
For hybrid sequencing of these samples, we recommend our “Hybrid; Illumina 400 Mbp + Nanopore 300 Mbp” service.
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.