The following guidance outlines some considerations and protocol modifications for the Monarch HMW DNA Extraction Kits when used upstream of Ultra Long (UL) DNA Sequencing with the Oxford Nanopore Technologies (ONT) workflow. Follow the standard protocol for the desired sample types, using the following recommendations and modifications:
- Cultured cells: Typically, the Monarch HMW kits yield more DNA than other commercial approaches. For transformed cell lines that contain additional copies of some chromosomes (e.g., HeLa, HEK293), yield can be up to 8-11 µg per 1 x 106 cells. In such cases, inputs of 4-5 x 106 cells are sufficient to reach the desired UL workflow input of 40 µg. If using more cells, consider using a portion of the eluate.
- Blood: The DNA amount present in blood is dependent on the leukocyte count, which is highly variable by donor. Blood samples with low leukocyte counts will yield ~ 25 µg DNA per ml of blood while samples with high leukocyte counts will give up to 65 µg DNA per ml of blood. Oxford Nanopore Technologies’ guidelines suggest a starting volume of 1.6 ml for cow blood, which typically has a lower leukocyte count than human blood. Given the variability, it is recommended to either do a pilot blood prep to reliably quantitate DNA content (500 µl blood sample lysed at 2000 rpm agitation speed), or to adjust the starting sample volume of the UL library prep so that 40 µg of DNA is used, by following the DNA measurement guidance referenced in “Quantitation Guidance” below.
- Tissue: When working with the Monarch HMW DNA Extraction Kit for Tissue (NEB #T3060), it may be necessary to combine 2 or 3 eluates from the same tissue material to reach the desired DNA input amount of 40 µg for the UL library prep.
Lysis Agitation Guidance:
- During lysis in the thermal mixer we recommend to use low agitation speeds. Agitation speeds of 300 - 800 rpm do not introduce detectable shearing based on pulsed field gel analysis. The optimum agitation speed may need to be optimized for each sample type, but for ultra-high molecular weight (UHMW) DNA isolated from different cell lines it was found that 600-700 rpm gave optimal UL sequencing results.
Washing and Drying Guidance:
- Do not allow the DNA to dry out while bound to the beads; add the next buffer immediately after removing the previous one and then proceed to the next sample. This is particularly important after the dry spinning step and the addition of the elution buffer.
- Keep the dry spinning pulse-spin as short as possible - the minifuge should not reach full speed.
- Work sample by sample, and add elution buffer immediately after the dry spin. For optimal result, pre-aliquot elution buffer in the 2 ml tube so that following the pulse spin, the beads can immediately poured into the elution buffer. The longer the DNA is left dry on the beads, the more difficult it will be to get it back into solution after elution.
- Use 200 µl of the Monarch Elution Buffer II for elution off of the beads.
- During the elution spin, centrifuge for 1 minute at 12,000 x g instead of 30 seconds.
- Before moving into the cleanup step in the library prep, UHMW DNA will need to be diluted to 750 µl with additional Monarch gDNA Elution Buffer II (10mM Tris, pH 9.0, 0.5 mM EDTA), (NEB #T3056).
- For accurate quantitation of UHMW DNA samples, please follow the guidance provided in the following publication: Koetsier PA, Cantor EJ. A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA. BioTechniques (2021). https://doi.org/10.2144/btn-2021-0051
- Monarch HMW DNA Extraction Kit for Cells & Blood Product Manual
- Monarch HMW DNA Extraction Kit for Tissue Product Manual
- Choosing Input Amounts for the Monarch HMW DNA Extraction Kits
- Choosing an Agitation Speed During Lysis with the Monarch HMW DNA Extraction Kits
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits