Protocol Guidance for Extraction of Ultra-High Molecular Weight (UHMW) Genomic DNA for Ultra-Long (UL) Read NGS Sequencing applications in Oxford Nanopore Technologies® workflows
The following guidance outlines considerations and protocol modifications for the Monarch HMW DNA Extraction Kits for Tissue (NEB #T3060) and Cells & Blood (NEB #T3050) when used upstream of Ultra Long (UL) DNA Sequencing* with the Oxford Nanopore Technologies (ONT) workflow (You may be required to log into the Nanopore Community for access.). Follow the standard protocol for the desired sample types (linked below), incorporating the modifications and guidance provided on this page.
- Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Cells (NEB #T3050)
- Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Bacteria (NEB #T3060)
- Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Tissue (NEB #T3060)
- Protocol for High Molecular Weight DNA (HMW DNA) Extraction from Blood (NEB #T3050)
Important Notes Before Extraction
- Prepare an elution buffer-Triton mix (also known as “EB+”) for the current experiment: Monarch Elution Buffer II + 0.02% Triton X-100 (e.g., add 20 µl of a 10% Triton X-100 solution to 10 ml Monarch Elution Buffer II. Mix thoroughly). At least 760 µl elution buffer-Triton mix is needed per library prep for PromethION (750 µl for library prep + 10 µl for quantitation) or 385 µl for a library prep that is run on MinION flow cells (375 µl +10 µl). Using this mix instead of Monarch Elution Buffer II results in better UL library prep and sequencing results.
- Monarch Elution Buffer II provided with the Monarch kit is supplied in volumes based on the standard extraction protocol which uses up to 200 µl per sample. As this protocol requires up to 760 µl per sample, additional Monarch Elution Buffer II may be required (NEB #T3056). This buffer contains 10 mM Tris-HCl, 0.5 mM EDTA pH 9.0. Triton X-100 should be added before use in the UL sequencing sample prep workflow as indicated above.
UHMW DNA Input for Library Prep:
- According to the guidance provided by ONT, the recommended input for the UL library prep is 40 µg when using PromethION flow cells. If working with the MinION flow cells, only 20 µg of DNA is needed. Sample input amounts for extraction should be chosen accordingly. Please note that the amount of DNA per µl of Fragmentation Mix (FRA) should be kept constant at 6-7 µg, which is the amount of DNA that can be isolated from 1x106 human cells; sample input amount should be adjusted when working with organisms of different sized genomes to maintain the same amount of isolated DNA.
- If working with MinION flow cells (which requires input of 20 µg) prior to library prep, UHMW DNA samples should be in 375 µl of EB+ (half of the 750 µl recommended by ONT). Thus, after elution in 200 µl, add 175 µl to create a total volume of 375 µl. All volumes in the library prep (ONT # SQK-ULK001) should also adjusted to 50% of the standard amounts.
Sample Input Guidance for Extraction
The guidance below is based on 40 ug input for PromethION flow cells. If working with MinION flow cells, reduce input accordingly (see above).
- Cultured cells: Typically, the Monarch HWM 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. Additionally, yields may also be somewhat higher than expected, as some cell lines have the tendency to clump, possibly resulting in an underestimation in the cell count. Hence inputs of 4-5 x 106 cells are often sufficient to reach the desired UL workflow input of 40 µg. If using more cells, quantitate yield2 and 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. The Oxford Nanopore Technologies (ONT) 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 for the UL library prep so that 40 µg of DNA is used, by following the DNA measurement guidance2.
- 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 on PromethION.
Direct Lysis for Cell & Blood Protocols
The standard workflow for the Monarch HMW DNA Extraction Kit for Cells & Blood (NEB #T3050) employs nuclei preparation, initially included for complete RNA removal. However, it has been observed that nuclei preparation may result in lower N50s in UL sequencing when compared with direct lysis. Moreover, the presence of RNA in UHMW DNA samples does not negatively affect UL sequencing. As such, a direct lysis approach, outlined below, can be employed as an alternative approach for improved UL sequencing results.
- Resuspend cells or leukocytes in 100 µl cold PBS.
- Create a premix of Direct Lysis Mix: for each sample, combine 100 µl Nuclei Prep Buffer, 100 µl Nuclei Lysis Buffer, and 10 µl Proteinase K. Vortex briefly to mix.
- Add 200 µl Direct Lysis Mix to resuspended cells and mix by inverting 10 times.
- Incubate for 10 minutes at 56°C with thermal mixer set at desired agitation speed (see below)
- Optional for cultured cells only: Following Proteinase K digestion, add 5 µl RNase A and mix by pipetting carefully 5 times using a wide-bore P1000 pipette tip. Incubate for 10 minutes at 56°C. This step is not necessary for blood samples, as leukocytes do not contain significant amounts of RNA at this stage. Due to high viscosity in cultured cell samples, RNA removal may be incomplete, leading to over-quantitation by spectrophotometer. Measurement with Qubit is recommended2.
Agitation Speed During Lysis:
For UL sequencing workflows, the ideal agitation speed during lysis may require optimization for each sample type but typically ranges from 0 up to 800 rpm. Agitation speeds up to 800 rpm do not introduce detectable shearing based on pulsed-field gel analysis. The authors of “FindingNemo: A Toolkit…”1 found 600-700 rpm to give optimal UL sequencing results in terms of N50 read length and data amount.
Binding & Elution Considerations:
Carry out the UHMW DNA binding incubation in the vertical rotating mixer for 8 minutes when working with 40 µg DNA, or 5 minutes when working with 20µg DNA or less. This ensures tight binging of the UHMW DNA to the glass beads and will prevent sample loss during the dry spin following the wash steps.
Alternative bead binding workflow
The following alternative bead binding workflow is an option for experienced users looking for improved sequencing metrics. Invert the sample via slow manual inversion (~5 seconds per full inversion). Stop inverting once the DNA forms a loose “jelly” around the beads and the viscosity of the solution has returned to normal levels. This usually takes 20-25 inversions for cell and blood samples, and ~40 inversions for tissue samples. By keeping the DNA in this loose form, it is less compact and will therefore go into solution easier at the end of the purification process, leading to better results during the library prep and sequencing. However, when removing the supernatant after bead binding, care should be taken not to remove part of the DNA jelly, as it can easily slip into the pipette tip. If necessary, leave some of the liquid behind.
Upon adding the wash buffer, the DNA “jelly” will contract tighter around the beads and the sample will be easier to handle. Do not spin the beads to dry them after the wash when using this alternative workflow, as some DNA may detach from the beads. Instead, pour the beads containing the loose DNA complex into the bead retainer and tap the bead retainer gently on absorbent paper to remove traces of wash buffer. The DNA will not be completely dried, but that is not necessary at this stage; it will lead to better solubility upon elution. Do not expose the DNA to air longer than necessary; after tapping, pour the beads immediately into the elution buffer EB+, and return the empty bead retainer into the collection tube. Spin briefly in a benchtop minifuge to remove traces of wash buffer from the bead retainer walls. The dried bead retainer can now be placed in the DNA low bind tube that is required for the elution step.
Using the elution buffer-Triton mix (EB+) instead of Monarch Elution Buffer II results in better UL library prep and sequencing results. Instructions for formulating this mix are provided in “Important Notes Before Extraction.”
After the washes and dry spinning step, the following steps should be employed for elution:
- Immediately pour the dried beads into the 2 ml tube with the pre-aliquoted elution buffer-Triton Mix (EB+).
- Incubate 10 minutes at 56°C to elute the DNA off the beads. To facilitate elution during incubation, gently pipette and dispense the eluate a few times over the glass beads using a wide bore pipette tip; ensure that the DNA has been released from the beads (the viscosity of the eluate will increase).
- If time is not limiting, let the eluate sit for at least 1 hour or overnight on the beads at room temperature before proceeding with the next step. This will improve elution efficiency and support effective resuspension of the UHMW DNA.
- Pour the beads with the elution buffer into the bead retainer sitting in a DNA low bind 1.5 ml tube.
- Centrifuge for 1 minute at maximum speed (>16,000 x g). Check if DNA was completely released from the beads. If after the spin DNA threads are visible between beads and eluate, centrifuge for 1 additional minute at max speed.
- Using a wide bore pipette tip, carefully pipette eluate up and down 5 times for homogenization and incubate the samples for 5-10 minutes at 37°C. If needed, repeat the careful pipetting several times to resuspend the DNA. Store at 4°C or continue with dilution and resuspension guidance below.
UHMW DNA Dilution & Resuspension for Library Preparation:
Samples should be further diluted to reach the optimal concentration for UL library preparation. Dilution should be done with the elution buffer-Triton mix that was used for elution, following the guidance below:
- Add 560 µl elution buffer-Triton mix (EB+) to the sample to bring the total volume to 760 µl. Use 185 µl if working with MinION flow cells, for a total volume of 385 µl.
- Pipette up and down 5-10 times using a P1000 wide-bore tip to resuspend the DNA.
- Incubate the samples for 5-10 minutes at 37°C.
- Repeat the steps 2 and 3 one or two times if samples do not appear homogeneous. Samples can be kept overnight at room temperature to support homogenization of the UHMW DNA or can be stored at 4°C. Once homogenized, take a 10 µl aliquot for quantitation2. Make sure DNA is well dissolved and no DNA clots are visible before proceeding with the Ultra Long library prep, Part 3 - Library Preparation. It is important to note that allowing the DNA at least 1 day to go into solution completely and “relax” typically leads to better library prep results.
Tips for Optimal Library Prep Results
- For maximal read lengths, it is essential to immediately and thoroughly mix the UHMW DNA with the diluted Fragmentation Mix (FRA). As an alternative to mixing via vortexing as described in the Oxford Nanopore Technologies library prep protocol, consider mixing with a wide bore pipette mixing ~20 times while keeping the tube on ice.
- The concentration of UHMW DNA used in the library prep is ~50 ng/µl. If the UHMW samples are further diluted to ~25 ng/µl, the viscosity is further reduced, usually resulting in increased efficiency of the library prep and therefore improved sequencing metrics. This is especially recommended when sequencing smaller DNA amounts on a MinION flow cell; dilute the isolated 20 µg of UHMW DNA (contained in 375 µl EB+) to a volume of 750 µl with EB+. When setting up the library, the amount of transposome remains constant; accordingly, add 3 µl FRA diluted in 244 µl FRA Dilution Buffer to 750 µl of 25 ng/µl DNA solution.
- Although cleaned up libraries can be run on the flow cell immediately after cleanup, optimal sequencing metrics (especially pore occupancy) are obtained if the library is given several hours, or ideally 1 day/overnight, at room temperature for complete resuspension.
- In our experience, higher N50 read lengths were obtained in some cases when less Fragmentation Mix (FRA) was used. The optimal amount of FRA may be slightly different for different sample types. If N50 read lengths are lower than expected, but the UHMW DNA is of high quality and has been measured accurately, consider optimizing the amount of FRA used.
- Cahyani, I., Tyson, J., Holmes, N., Quick, J., Loman, N., Loose, M. “Finding Nemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms” Protocols.io, (09/2021) https://dx.doi.org/10.17504/protocols.io.bxwrppd6
- Koetsier PA, Cantor EJ. A simple approach for effective shearing and reliable concentration measurement of ultra-high-molecular-weight DNA. BioTechniques 71 (2), 439-444 (2021). https://www.future-science.com/doi/10.2144/btn-2021-0051
- Protocol for UHMW DNA Cleanup in the Oxford Nanopore Technologies® UL Library Prep Workflow
- Monarch HMW DNA Extraction Kit for Cells & Blood Product Manual
- Monarch HMW DNA Extraction Kit for Tissue Product Manual
- Homogenization of High Molecular Weight DNA (HMW DNA) Samples
- Measuring, Analyzing & Storing High Molecular Weight DNA (HMW DNA) Samples
- Considerations and Performance Data for Nanopore Sequencing of High Molecular Weight DNA (HMW DNA)
- Troubleshooting Guide for High Molecular Weight DNA Extraction Using the Monarch HMW DNA Extraction Kits