Sanger Tree of Life HMW DNA Extraction: Manual Plant MagAttract v.2/3
Caroline Howard, graeme oatley, Maja Todorovic, Amy Denton
HMW DNA extraction
magnetic bead extraction
manual DNA extraction
MagAttract
Plant HWM DNA extraction
solid phase reversible immobilisation
reference genome
long read sequencing
Abstract
This protocol describes the manual extraction of HMW DNA from plant or fungi tissue samples from a variety of species intended for long-read sequencing. It employs the Qiagen MagAttract HMW DNA extraction kit. This process is effective for approximately 60% of the plant species covered by the Tree of Life Programme, but the resulting yield of CCS data from PacBio sequencing was more stable than with the v.1 protocol. This protocol is particularly useful for samples with limited tissue availability, as it ensures the maximum amount of HMW DNA can be extracted and recovered. The output of this protocol is HMW DNA, which depending upon yield and genome size of the species, can be directed towards either HMW DNA Pooling, HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI HiFi, HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. This protocol was adapted from Sanger Tree of Life HMW DNA Extraction: Manual Plant MagAttract v.1 to improve sample lysis (v.2) and include a pre-shear SPRI of the HMW DNA extracted (v.3), and has since been updated to Sanger Tree of Life HMW DNA Extraction: Manual MagAttract v.4 to further improve sample lysis.
Acronyms
HMW: high molecular weight
SPRI: solid-phase reversible immobilisation
HiFi: high fidelity
LI: low input
ULI: ultra-low input
CCS: circular consensus sequencing
Before start
Add 100% ethanol to the MW1 and PE wash buffers as per manufacturer’s instructions.* Set one heat block to 55 °C and another to 25 °C.
Steps
Sample lysis
Prepare a lysis buffer master mix:
| A | B |
|---|---|
| Phosphate-buffered saline (PBS) | 200 µL |
| Proteinase K | 20 µL |
| RNase A | 4 µL |
| Buffer AL | 150 µL |
Transfer 50 mg of cryogenically disrupted plant/fungi tissue from each sample to 2 mL microcentrifuge tubes and place on dry ice to keep the samples frozen.
Add 374 µL of the lysis buffer master mix to each sample, then homogenise sample and mastermix by gently pipetting 10 times with a wide-bore pipette tip.
Centrifuge tube briefly to collect in a mini-centrifuge, then incubate on the heat block at 55 °C at 600 rpm for 1 hour.
DNA isolation
Once samples have completed lysing, remove sample tubes from the heat block and briefly centrifuge in a mini-centrifuge to spin down.
Using a wide-bore pipette tip, set the volume to 380 µL, transfer lysate to individual microcentrifuge tubes, whilst avoiding insoluble material.
Add 280 µL Buffer MB to each sample and 15 µL of Suspension G beads. Invert the tube 10–20 times to ensure the beads are suspended in the lysate. Allow 5 minutes for binding.
Briefly centrifuge the samples in a mini-centrifuge to collect at the bottom of the tube.
Place the tubes on the magnetic rack and allow 2–5 minutes for the beads to migrate (more viscous samples will take longer). Remove the supernatant and discard.
Remove the tubes from the magnetic rack and add 700 µL Buffer MW1 directly to the bead pellet, then invert the tube 10–20 times to ensure the beads are suspended in the lysate.
Place the tubes on the magnetic rack and allow 2-5 minutes for the beads to migrate (more viscous samples will take longer). Remove the supernatant and discard.
Repeat the MW1 wash for a total of two washes (steps 10 and 11).
Remove the tubes from the magnetic rack and add 700 µL Buffer PE directly to the bead pellet and invert 10–20 times to resuspend the beads.
Place the tubes on the magnetic rack and allow 2–5 minutes for the beads to migrate (more viscous samples will take longer). Remove the supernatant and discard.
Repeat the PE wash for a total of two washes (steps 13 and 14).
Briefly centrifuge the tubes in a mini-centrifuge and place the sample back on the magnetic rack. Use a small micropipette to remove any residual wash buffer.
Pipette 700 µL nuclease-free water onto the side opposite of the beads in the microcentrifuge tubes whilst the tubes are on the magnetic rack. Do not pipette the nuclease-free water directly onto the bead pellet. Incubate for exactly 1 minute then slowly aspirate and discard water from the tubes.
Repeat step 17 for a total of two washes.
Remove the samples from the magnetic rack and add 200 µL of Buffer AE directly to the bead pellet. Mix, either by gently flick mixing or using a wide-bore pipette tip in order to dislodge the pellet from the tube.
Incubate for 15 minutes at room temperature, with a gentle mix halfway through and again at the end.
Briefly centrifuge (spin down) the sample in a mini-centrifuge and place on a magnetic rack, allowing 2–5 minutes for bead migration.
Using a 200 μL wide-bore pipette tip, carefully transfer the supernatant containing purified gDNA to a fresh microcentrifuge tube.
Remove the sample from the magnetic rack. Add 200 μL Buffer AE to the bead pellet. Incubate at 25 °C, shaking at 1000 rpm, for three minutes.
Centrifuge the tube briefly in a mini-centrifuge and place it on a magnetic rack for 2–5 minutes for the beads to migrate.
Using a wide-bore pipette tip, carefully transfer the supernatant containing purified gDNA to the same microcentrifuge tube as step 22.
Store the extracted gDNA sample at 4 °C.
