TITLE: Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.4
AUTHORS: Benjamin Jackson, Caroline Howard

[DESCRIPTION]
This protocol describes the automated extraction and SPRI of HMW DNA from cryogenically homogenised or bead-beaten tissue samples from plants and fungi intended for long-read sequencing. It employs the Qiagen MagAttract HMW DNA extraction kit and the Thermo Fisher KingFisher™ Apex. This process is effective for a wide variety of plant species covered by the Tree of Life Programme. 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 PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. This protocol was adapted from Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.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
LI: low input
ULI: ultra-low input

[BEFORE_START]
Add 100% ethanol to the MW1 and PE wash buffers as per manufacturer’s instructions.
AMPure PB beads are stored in the fridge at 4 °C – take them out 30 minutes before starting the 0.45X SPRI KingFisher™ Apex protocol to bring them to room temperature. 

[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.
For the 0.45X SPRI, the DNA and AMPure beads should not sit together in the sample plate for more than 5 minutes before starting the KingFisher™ Apex.
An experienced operator can expect to comfortably process up to 32 samples, with approximately 2–3 hours handling time over a start to finish period of 4-5 hours. This estimation includes the utilisation of the KingFisher™ Apex for both the extraction and SPRI protocols, and excludes subsequent QC checks.

Additional Notes:
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.
Both the KingFisher™ Apex protocol scripts and the KFX.files have been made available for this protocol – the KFX.files require ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocols to be viewed on a PC or laptop. Alternatively, the files can be transferred directly onto a KingFisher™ Apex instrument using a USB flash drive.

[STEPS]
SECTION: Sample lysis
1. Set one heat block with a 50 mL SmartBlock  to 65 °C and another heat block with a 2 mL SmartBlock to 55 °C.
 
  ReagentVolume per samplePhosphate buffered solution (PBS)200 µLBuffer AL150 µL

SECTION: Sample lysis
2. Prepare a lysis buffer master mix in a 50 mL centrifuge tube:
SECTION: Sample lysis
3. 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.
SECTION: Sample lysis
4. 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.
SECTION: Sample lysis
5. Transfer the samples to a pre-chilled cold block on wet ice and incubate for 10 minutes to equilibrate temperature.
SECTION: Sample lysis
6. 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.
SECTION: Sample lysis
7. 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. 
SECTION: Sample lysis
8. After 5 minutes incubation, resuspend any severely aggregated samples by pipette mixing with a wide-bore pipette tip.
SECTION: Sample lysis
9. 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.
SECTION: Sample lysis
10. Incubate samples for a further 45 minutes on the heat block at 55 °C at 600 rpm.
SECTION: Sample lysis
11. 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.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
13. 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.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
14. Centrifuge the samples for 10 minutes at 8,000 rpm at room temperature.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
15. Using a wide-bore pipette tip, set the volume to 380 µL, gently transfer lysate from the sample tubes to individual wells in the sample plate, taking care to avoid aspirating the pelleted tissue particles.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
16. Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex DNA Extraction Protocol Script/attached KFX file in the Materials section) and select using the play button.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
17. Load the filled plates onto the instrument following the instructions provided on screen.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
18. Prior to loading the “Sample Plate”, the instrument will prompt to remove the “Tip Plate”. Once the final plate is loaded, the protocol will automatically begin; this takes approximately 50 minutes.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
19. Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
20. Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the DNA using a wide-bore pipette tip.
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
22. Set-up the KingFisher plates for the 0.45X SPRI as detailed below:

   PlatePlate TypeReagent(s) requiredTip Plate1 mL Deep-well96-well tip comb (no reagent)Sample Plate (Elution Plate 1 from DNA Extraction Protocol)1 mL Deep-well380 µL DNA + 171 µL AMPure PB beadsEthanol Wash Plate1 mL Deep-well1000 µL 80% EtOH (freshly made)Elution Plate200 µL standard135 µL Buffer EB


SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
21. Using a 200 µL multi-channel pipette and wide-bore tips, pipette eluates from Elution Plate 2 into Elution Plate 1, and gently pipette mix 5–10 times with wide-bore tips to fully homogenise DNA in the eluate. Elution Plate 1 with the combined eluates is now the ‘Sample Plate’ for the 0.45X SPRI.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
12. Whilst samples lyse, label nine 1 mL 96-well deep-well KingFisher™ plates and fill the number of wells required for the number of samples in each plate as follows:
 
  Tip plate96-well tip comb (no reagent)Elution 2 200 µL Buffer AE Elution 1 200 µL Buffer AE NFW Wash 500 µL Nuclease-Free Water PE Wash 2700 µL Buffer PEPE Wash 1700 µL Buffer PEMW1 Wash 2700 µL Buffer MW1MW1 Wash 1700 µL Buffer MW1Sample plate15 µL Suspension G magnetic beads 280 µL Buffer MB

23. Select the required 0.45X SPRI protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex 0.45X SPRI Protocol Script/attached KFX file in the Material section) and select using the play button.
24. Load the filled plates onto the instrument following the instructions provided on screen.
25. Once the final plate is loaded, the protocol will automatically begin; this will take approximately 40 minutes.
26. Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
27. Using a wide-bore pipette tip, transfer the 130 µL of eluate from the elution plate into microcentrifuge tubes. 
28. Incubate the DNA at room temperature overnight and perform the required QC the following morning.
29. Store the DNA at 4 °C.