Standalone ONT Ligation Sequencing

Eurofins Genomics offers ligation sequencing using Oxford Nanopore Technology (ONT). Ligation sequencing is a popular method for validating library preparation protocols that utilizes a native barcode kit, achieving a median raw read accuracy of Q20+ (99%).

Eurofins Genomics offers standalone ONT ligation library preparation and sequencing services. Nanopore sequencing alone can be a cost-effective option for verifying structural modifications, such as large insertions, though higher sequencing depth may be necessary to confirm consensus sequences at the per-base level. These methods provide average quality scores of Q30 (99.9%+) or higher, making them more suitable for verifying single nucleotide changes.

In addition, ONT ligation sequencing is especially helpful for base modifications.


Pricing

Description Catalog # Estimated data output Price per sample Turnaround time
Ligation Sequencing - Medium 3010-ONTLSM 200 Mbp Long Reads $240 3 business days
Ligation Sequencing - Large 3010-ONTLSL 400 Mbp Long Reads $315 3 business days
Ligation Sequencing - XL 3010-ONTLSXL Min 900 Mbp Long Reads $420 3 business days

 

 

Specifications

The nanopore platform requires a high input of DNA. For hybrid sequencing with our Nanopore Combo services, two separate DNA aliquots are needed—one for each sequencing pipeline. To ensure accurate concentration measurements, we highly recommend using a dsDNA-specific, dye-based quantification method, such as Qubit or PicoGreen.

  • Total volume of 60 µL or more
  • Eluted into water or dilute Tris/TE buffer
  • Sample concentration greater than: 100 ng/µL

Maximizing DNA Fragment Length for Optimal Long-Read Sequencing

To enhance the performance of long-read sequencing platforms, it is essential to maximize DNA fragment lengths during extraction and handling.

The DNA extraction process is often the primary factor influencing fragment length. Gentle extraction methods, such as enzyme-based kits, typically yield the longest fragments. However, cells that are difficult to lyse may require physical or thermal lysis techniques, which can result in shorter DNA fragments. Therefore, it is crucial to fine-tune extraction methods for specific sample types to achieve the optimal balance.

Eurofins Genomics is committed to sequencing all submitted samples and does not impose a minimum length requirement. We advise our customers to assess fragment length prior to submission to ensure the best possible results.

DNA integrity can also be affected by handling and storage conditions, particularly during shipping. To minimize degradation, it is advisable to avoid freeze-thaw cycles and other extreme changes. For frozen DNA samples, we recommend shipping on dry ice, ensuring an adequate supply (5-10 lbs per day of expected transit). Liquid samples should be shipped on ice packs to buffer against temperature fluctuations, especially during the summer months.

Adhering to these guidelines will help maintain DNA quality and optimize the performance of long-read sequencing technologies.

 

The ligation sequencing library preparation protocol method circumvents the need for PCR, fragmentation, and size selection. The advantages of this approach are substantial, including the elimination of PCR-induced errors and enzyme bias. Furthermore, nanopore sequencing allows for the direct analysis of native DNA molecules, enabling the generation of exceptionally long reads and providing kinetic data that can reveal base modifications in nucleic acids.

However, the sequencing of native molecules necessitates a higher input compared to PCR-based methods, primarily due to the lower efficiency of ligation-based preparation. This inefficiency is exacerbated by the absence of amplification. Additionally, the nanopore platform's sensitivity to certain contaminants, such as polysaccharides found in some fungi, plants, and insects, as well as excessive unprocessed nucleic acids (e.g., surplus RNA in a DNA library), can pose challenges during extraction.

 

ONT sequencing provides kinetic data for each native DNA molecule as it moves through a nanopore, by measuring the electrical current across the nanopore. Variations in this current are analyzed not only for base calling but also for detecting nucleotide modifications. Currently, 5mC metadata is generated during the basecalling process, stored in the resulting BAM files, and can be utilized later to identify 5mC methylation sites using specialized tools.

While ONT itself offers a limited set of tools for this analysis, such as Remora and Dorado, there is a thriving community of researchers dedicated to developing new methods for predicting various nucleotide modifications.

 

Eurofins Genomics is dedicated to making Nanopore sequencing accessible and cost-effective for a variety of projects, including those involving small genomes. By multiplexing our single sample packages, we can optimize the output and cost-efficiency of Nanopore flowcells for both hybrid sequencing and ONT-exclusive services. This approach ensures that high-quality sequencing data is available to projects of all sizes.

“Your sequencing reactions were perhaps the turning point in my work. Obtaining such good quality reads is rare. I have submitted many samples but this is was the best, we should explore all variable associated with the success to make it reproducible, quality, quantity of DNA to reagents used at Eurofins.”

Shyam G

Shyam G

Customer

“We got the sequences back and are quite happy with the quality. One of the orders had amplicons that we have been sequencing routinely for many years and quality from Eurofins seems to be better. Also, our overall experience with Eurofins website, sample submission, email notifications and sequencing results was positive and user friendly. We are looking forward to sequencing with Eurofins.”

Anna M.

Anna M.

Customer

“This was really impressive. Cannot believe we can get full plasmid sequencing for $15 a pop. Greatly appreciated.”

Drake R. PhD

Drake R. PhD

Customer

“Whole Plasmid Sequencing is good service at a great price point that saves me time. A great addition to the sequencing toolkit that I will be using more in the future.”

Shane A

Shane A

Customer

“Beautiful sequences”

Dr. Dorothy E.

Dr. Dorothy E.

Customer

“Speed of analysis is exceptional!”

Adeleye A.

Adeleye A.

Customer

“I have to say, your sequencing is outstanding. From ease of drop off to rapid turn around, to your customer support people if I do have a question and call. I refer everyone to Eurofins. Thanks for doing a great job!”

Amy H.

Amy H.

Customer

“Incredibly fast turnaround, and the sequences are stellar. I’m really thankful for your services, they make my job much easier.”

Joseph D.

Joseph D.

Customer

“I am consistently surprised and pleased with how quickly you can provide sequencing data! I dropped my samples off late last evening at my local dropbox and my results were waiting in my inbox when I arrived at work this morning! My PI and I were just joking that we make a comment every time about how the results come so quickly. Maybe we just aren't used to such good customer service. Great job.”

Eva J.

Eva J.

Customer

“Love your service. You guys are my go to service, and I constantly recommend you to local folks who do sequencing.”

Daniel K

Daniel K

Customer

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