Sanger Tree of Life HMW DNA Extraction: Manual Plant MagAttract v.4
Caroline Howard, Benjamin Jackson
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, using 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, and the resulting yield of CCS data from PacBio sequencing has been good. 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.2/3 to further improve sample lysis. These improvements have been made through a combination of preheating the lysis buffer, delaying the addition of RNase A to later on in sample lysis and increasing centrifugation of the lysate, which have led to a reduction in tissue clumping, minimised oxidative damage of the DNA, reduced Proteinase K inhibition of RNase A and increased purity of the HMW DNA due to the exclusion of aggregated, insoluble or sedimented contaminants.
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 with a 50 mL SmartBlock to 65 °C, another heat block with a 2 mL SmartBlock to 55 °C and a third heat block with a 2 mL SmartBlock to 25 °C.
Steps
Sample lysis
Prepare a lysis buffer master mix in a 50 mL centrifuge tube:
| A | B |
|---|---|
| Phosphate-buffered saline (PBS) | 200 µL |
| Buffer AL | 150 µL |
Place the lysis buffer on the 65 °C heat block and incubate at 400 rpm for at least 20 minutes. Keep at temperature until added to the sample.
Transfer 50 mg of cryogenically disrupted tissue from each sample to 2 mL microcentrifuge tubes.
- Ensure the disrupted tissue is completely disrupted into a fine powder; avoid matted/clumped powder. This is crucial for optimal DNA yield and integrity; poorly disrupted tissue drastically decreases lysis and extraction efficiency.
- Any samples containing poorly disrupted tissue ‘chunks’ should be flagged as requiring reprocessing and further cryogenically disrupted.
Transfer the samples to a pre-chilled cold block on wet ice and incubate for 10 minutes to equilibrate temperature.
Add 20 µL Proteinase K (for n+1 samples) to the preheated lysis buffer immediately prior to initiating lysis, swirling the centrifuge tube to mix.
Add 370 µL of the preheated lysis buffer plus Proteinase K to each sample, immediately homogenising the lysate by mixing with 5 rapid pulse vortexes, and place on the 55 °C heat block at 600 rpm for 15 minutes.
After 5 minutes incubation, resuspend any severely aggregated samples by pipette-mixing with a wide-bore pipette tip.
After the initial 15 minute incubation, add 4 µL RNase A to each sample and mix thoroughly by inversion until any aggregated, insoluble or sedimented tissue particles are resuspended.
Incubate samples for a further 45 minutes on the heat block at 55 °C at 600 rpm.
During this incubation, samples should be occasionally mixed (every 5–15 minutes) by inversion to resuspend sedimented particles. Do not mix the samples by inversion for the final 15 minutes of lysis, allowing aggregated, insoluble or sedimented tissue particles to settle at the bottom of the tube.
DNA isolation
Once samples have completed lysing, remove sample tubes from the heat block and allow the lysate to settle to the bottom of the tube for 5 minutes.
Centrifuge the samples for 10 minutes, 8,000 rpm at room temperature.
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 17 and 18).
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 20 and 21).
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 24 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 1,000 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 29.
Store the extracted gDNA sample at 4 °C.
