TITLE: Sanger Tree of Life HMW DNA Extraction: Manual Plant MagAttract v.2/3
AUTHORS: Maja Todorovic, graeme oatley, Amy Denton, Caroline Howard

[DESCRIPTION]
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.

[GUIDELINES]
For the lysis buffer master mix, prepare enough for n+1 samples to allow for pipetting errors.
Keep samples on dry ice to maintain temperature and prevent nucleic acid degradation until the lysis buffer is ready to be added to them.
An experienced operator can expect to comfortably process 8 samples, with approximately 2 hours handling time over a start to finish period of 4 hours. This estimation excludes subsequent QC checks.

Additional Notes:
This protocol contains the sample lysis procedure for both Plant MagAttract v.2 and Plant MagAttract v.3: 
For samples that are suitable for LI sequencing on HiFi, the extracts produced at the end of this protocol should be directed towards HMW DNA Fragmentation: Diagenode Megaruptor® 3 for LI HiFi - this is Plant MagAttract v.2. 
For samples that are suitable for LI sequencing on PacBio, the extracts produced at the end of this protocol can undergo a 0.45X SPRI, using either the KingFisher™ Apex 0.45X SPRI Protocol detailed in the Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.3, or following the Sanger Tree of Life Fragmented DNA clean up: Manual SPRI protocol using a bead:sample ratio of 0.45:1 – this is Plant MagAttract v.3.
FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for DNA storage, all routine DNA extracts are stored in FluidX tubes.

[STEPS]
SECTION: Sample lysis
1. Prepare a lysis buffer master mix:
  Phosphate buffered solution (PBS)200 µLProteinase K20 µLRNase A4 µLBuffer AL150 µL

SECTION: Sample lysis
2. 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.
SECTION: Sample lysis
3. 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.
SECTION: Sample lysis
4. Centrifuge tube briefly to collect in a mini-centrifuge, then incubate on the heat block at 55 °C at 
600 rpm for 1 hour.
SECTION: DNA isolation
5. Once samples have completed lysing, remove sample tubes from the heat block and briefly centrifuge in a mini-centrifuge to spin down.
SECTION: DNA isolation
6. Using a wide-bore pipette tip, set the volume to 380 µL, transfer lysate to individual microcentrifuge tubes, whilst avoiding insoluble material.
SECTION: DNA isolation
7. 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.
SECTION: DNA isolation
8. Briefly centrifuge the samples in a mini-centrifuge to collect at the bottom of the tube.
SECTION: DNA isolation
9. 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.
SECTION: DNA isolation
10. 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.
SECTION: DNA isolation
11. 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.
SECTION: DNA isolation
12. Repeat the MW1 wash for a total of two washes (steps 10 and 11).
SECTION: DNA isolation
13. 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.
SECTION: DNA isolation
14. 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.
SECTION: DNA isolation
15. Repeat the PE wash for a total of two washes (steps 13 and 14).
SECTION: DNA isolation
16. 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.
SECTION: DNA isolation
17. 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.
SECTION: DNA isolation
19. 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.
SECTION: DNA isolation
20. Incubate for 15 minutes at room temperature, with a gentle mix halfway through and again at the end.
SECTION: DNA isolation
21. Briefly centrifuge (spin down) the sample in a mini-centrifuge and place on a magnetic rack, allowing 2–5 minutes for bead migration.
SECTION: DNA isolation
22. Using a 200 μL wide-bore pipette tip, carefully transfer the supernatant containing purified gDNA to a fresh microcentrifuge tube.
SECTION: DNA isolation
23. 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.
SECTION: DNA isolation
24. Centrifuge the tube briefly in a mini-centrifuge and place it on a magnetic rack for 2–5 minutes for the beads to migrate. 
SECTION: DNA isolation
25. Using a wide-bore pipette tip, carefully transfer the supernatant containing purified gDNA to the same microcentrifuge tube as step 22.
SECTION: DNA isolation
26. Store the extracted gDNA sample at 4 °C.
SECTION: DNA isolation
18. Repeat step 17 for a total of two washes.