Ligation Sequencing

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. If there is insufficient material for the ONT portion, the hybrid package will be converted to the corresponding Illumina package, and only Illumina data will be provided.

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

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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.