We recommend: New England Biolabs Monarch® Spin gDNA Extraction Kit, QIAGEN Genomic-tip-500/G, QIAGEN MagAttract HMW DNA Kit, QIAGEN Puregene Yeast/Bacteria Kit. In general, all extraction methods that allow the generation of HMW gDNA fulfilling the above requirements can be used. To ensure to extract HMW DNA we recommend

• Wash bacterial cell pellets before DNA extraction with PBS to remove potential inhibitors
• Add 1ml of PBS and resuspend cells by pipetting
• Pellet the cells by centrifugation and discard the supernatant
• Avoid vortexing and fast or unnecessary pipetting; use wide-bore tips only
• Elute in nuclease-free elution buffer, not water
• Avoid over-drying of gDNA
• Do not expose DNA to high temperatures (>37°C) for >1 hour
• Use buffer with appropriate pH 7.5- 8.5
• Avoid intercalating fluorescent dyes, or UV radiation
• Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months

Short DNA fragments are typically not ideal for long-read sequencing, as they can result in poor assembly quality. We recommend that at least 50% of the DNA is above 15 kb in length. To improve sequencing outcomes, we recommend removing shorter DNA fragments from your sample before submission. One effective protocol involves using 4X diluted SPRISelect beads (35% volume by volume) to eliminate fragments smaller than 3-4 kb.

Please note that the DNA quantity will decrease following this process, so it’s essential to quantify the remaining DNA to ensure sufficient material is available for sequencing.

• Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
• Dilute SPRISelect beads with Elution Buffer to 35% (volume by volume).
• Resuspend the diluted SPRISelect beads by vortexing.
• Add 4x volume of the resuspended diluted SPRISelect Beads to each gDNA and mix by flicking the tube.
• Incubate for 5 minutes at room temperature.
• Prepare sufficient fresh 80% EtOH in nuclease-free water for all of your samples.
• Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
• Keep the tube on the magnet and wash the beads with freshly prepared 80% EtOH without disturbing the pellet. The EtOH volume must be enough to cover the beads entirely. Remove the EtOH using a pipette and discard.
• Repeat the previous step.
• Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual EtOH. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
• Remove the tubes from the magnetic rack and resuspend the pellet in e.g. 50 µL nuclease-free 1xTE or EB. Spin down and incubate for 10 minutes at 37°C with gentle agitation (700rpm) on a shaker.
• Pellet the beads on a magnet until the eluate is clear and colourless.
• Remove and retain the eluate into a clean 1.5 ml Eppendorf Safe Lock Tubes™

Alternatively, we recommend the DNA clean-up and size selection for long-read sequencing from Jones et al. 2021 (Jones A, Torkel C, Stanley D, Nasim J, Borevitz J, et al. (2021) High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. PLOS ONE 16(7): e0253830. https://doi.org/10.1371/journal.pone.0253830; https://www.protocols.io/view/dna-clean-up-and-size-selection-for-long-read-sequ-261ge6ymol47/v4)

Purity:

• Phenol, chloroform and other related reagents will inhibit the library preparation and must be completely removed. Although spectrophotometric measurements may not detect all types of contamination, we highly recommend performing them to assess sample purity. Ideally, the 260/280 ratio should be above 1.8, and the 260/230 ratio should fall between 2.0 and 2.2. If the sample is contaminated and does not meet this metric, either re-extract the sample or clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.Must not contain RNA; we strongly recommend RNase treatment during extraction
• Must not contain denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
• Must not contain residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.)
• Must not contain insoluble material or be colored or cloudy