TITLE:  Sanger Tree of Life HMW DNA Extraction: Automated Plant Organic HMW gDNA Extraction (POE)
AUTHORS: Benjamin Jackson, Caroline Howard

[DESCRIPTION]
The Plant Organic HMW gDNA Extraction (POE) protocol acts as the Sanger Tree of Life's programmes mid-throughput, reserve gDNA extraction procedure for all plant species too recalcitrant to yield HMW gDNA of adequate quality or quantity with the Plant MagAttract v.4 protocol. Developed in-house, the POE protocol is highly efficient at isolating pure, high-quality and high molecular weight (HMW) gDNA from the majority of plant species to an extent adequate for long-read sequencing.

The POE protocol is divided into four stages: (1) direct lysis of tissue homogenates with an SDS-based buffer containing reducing agents to mitigate oxidative DNA damage, (2) centrifugation and KDS-protein complex precipitation using potassium acetate, (3) gDNA isolation by two chloroform phase separations, (4) and gDNA capture/purification using a 1X Sera-Mag™ SpeedBead and 0.45X AMPure® PB double SPRI cleanup/size selection. Outcomes from 65–100 mg of fresh-frozen tissue homogenates are generally sufficient quantities (1-20 μg) of high purity, ultra HMW (uHMW; 100 kb+) gDNA adequate for multiple high-quality long-read sequencing events. However, outcome success is dependent on the plant species, tissue type and sample quality used.

The output of this protocol is uHMW gDNA, which depending upon yield and genome size of the species can be directed downstream towards HMW DNA Pooling, HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio.

Acronyms
HMW: high molecular weight
uHMW: ultra high molecular weight
gDNA: genomic DNA
SPRI: solid-phase reversible immobilisation
LI: low input
ULI: ultra-low input

[BEFORE_START]
Ensure all appropriate reagents in the 'materials' section have been prepared before starting the protocol:

500 μL of Sera-Mag™ SpeedBead solution is needed per sample; this requires the preparation of 50% PEG 8000, 10% Tween-20, SpeedBead wash suspension and SpeedBead binding solution before starting the protocol.

Prepare the 1M Tris base solution (pH 8.0) and potassium acetate solution (KAc; pH 7.4) before starting the protocol.

[GUIDELINES]
High quality, young leaf material with no observable damage, disease or other stressors, of which generally have less contaminant accumulation and are most pliable, should be preferentially selected for optimal outcomes downstream.

Alternative pliable tissue types (e.g. herbaceous stem, petiole or flower) may be selected when required.

Recalcitrant sample types (e.g. woody or rigid, fibrous tissues) or tissues with signs of stress can be selected, but results may vary.


Plant tissues should be preserved via flash freezing and stored under constant cryogenic conditions thereafter (e.g. on dry ice or within a –80 °C freezer). Alternative preservation techniques can be performed but outcomes may vary.

Whole-frozen or disrupted plant tissues can be stored long term at –80 °C with minimal detrimental effects observed.

Premature thawing of material or repeat freeze-thaw cycles can drastically decrease extraction efficiency due to gDNA degradation.


Ensure plant tissue is completely disrupted into a fine powder; avoid matted/clumped powder or fibrous material. Increased quality tissue homogenates result in increased quality and quantities of HMW gDNA.

Complete disruption is crucial to ensure optimal DNA yield and integrity; poorly disrupted input material can drastically decrease extraction efficiency and all quantifiable outcomes.

Plant tissues can be disrupted via cryogenic grinding (mortar and pestle), with the CP02 cryoPREP Automated Dry Pulverizer, or by cryogenic bead beating – it is recommended to follow the standardised steps outlined in either the 'Sanger Tree of Life Sample Homogenisation: Cryogenic Bead Beating of Plants with FastPrep-96 protocols' or 'Sanger Tree of Life Sample Homogenisation: Covaris cryoPREP® Automated Dry Pulverizer' protocols.


An experienced operator of the POE protocol can expect to comfortably process up to 24 samples per session, with 2–3 hours of handling time required over a start to finish period of 4–6 hours. This estimation excludes the overnight incubation of eluates to solubilise gDNA, subsequent QC checks, and includes the utilisation of the KingFisher™ Apex Instrument to perform the double SPRI cleanup/size selection – alternatively, this can be performed manually, which can be viewed in the manual version of this protocol.


Additional Notes
It is recommended to split larger numbers of samples (12+) into 2 batches during phase separation, starting step 8 on the second batch once the first is almost ready to come off the tube rotator (step 8.2).

Tri-coded FluidX tube are used throughout the Tree of Life programme in order to track samples, therefore all routine DNA extracts are stored in FluidX tubes. 

Both the KingFisher™ Apex protocol script and the KFX.file have been made available for this protocol – the KFX.file requires ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocol to be viewed on a PC or laptop. Alternatively, the file can be transferred directly onto a KingFisher™ Apex instrument using a USB.

Whilst highly effective at isolating gDNA from endogenous contaminants, this protocol is generally unsuitable for species with exceptionally high polysaccharide concentrations that form viscous lysates. For these, a pre-lysis hypertonic sorbitol wash of tissue homogenates should be performed, this will be included as an optional step in subsequent versions of the POE protocol.

[STEPS]
SECTION: Sample lysis
1. Prepare an adequate volume of the ‘Direct Plant Lysis Buffer’ (recipe in Materials).

Preheat the direct plant lysis buffer for 15–30 mins, 65 °C at 400 rpm prior to use, ensuring that all reagents are completely dissolved. 

Add DTT and Proteinase K to the direct plant lysis buffer immediately prior to use, ensuring both reagents are thoroughly mixed.
SECTION: Sample lysis
2. Aliquot 65–100 mg of cryogenically disrupted tissue samples into individual 2 mL Lo-Bind tubes on dry ice.

Lower than recommended quantities of tissue homogenate (down to >15 mg) can be used, but gDNA yield may vary.
SECTION: Sample lysis
3. Transfer the 2 mL Lo-Bind tubes containing sample to wet ice for 10 minutes, allowing sample temperature to equilibrate.

SECTION: Sample lysis
4. Perform the direct sample lysis.
SECTION: Sample lysis
4.1. Add 550 μL of preheated direct plant lysis buffer (65 ºC) to the first sample - immediately pulse vortex 5 times at full speed until homogenous, and place on a heat block at 55 ºC, 600 rpm. Repeat for each sample.

Some samples will not homogenise through pulse-vortexing. It is recommended that these samples are continuously vortexed for 5 seconds. If this does not homogenise the sample either, proceed to step 4.2.
SECTION: Sample lysis
4.2. Once all samples are homogenised and have begun incubation, inspect each by inverting to mix. Any samples with aggregated tissue that can’t be homogenised through inversion should be thoroughly mixed with a wide bore P1000 tip until clumps are separated and the sample is homogeneous.
SECTION: Sample lysis
4.3. After 15 minutes of incubation, add 4 µL RNase A to each sample and mix by pulse-vortexing 5 times until any aggregated, insoluble or sedimented tissue particles are resuspended. Repeat step 4.2 for samples that have reaggregated and remain clumped after vortexing.

The 4 µL RNase A can be diluted in 6 µL PBS per sample and added to the sample with a Multipette to improve ergonomics.
SECTION: Sample lysis
4.4. Incubate for another 45 minutes, 55 °C at 600 rpm.

Samples can be routinely resuspended by inversion, or pipette-mixed with a wide bore P1000 tip to remove aggregates, to improve lysis performance. However, this is not essential.

Do not agitate the samples by mixing for the last 15 minutes of lysis; allow any unlysed sediment to settle at the bottom of the tube.
SECTION: Sample lysis
4.5. Whilst the samples are incubating, prepare a fresh 2 mL Lo-Bind tubes containing 150 μL of cold potassium acetate solution (4 °C; 5 M; pH 7.4) for each sample, and incubate on wet ice until temperature equilibrates.
SECTION: Sample lysis
5. Remove the samples from the heat block, allow the lysate to briefly settle (1 to 5 minutes), and then centrifuge for 10 minutes, 8,000 rpm at room temperature.

Avoid disturbing the insoluble sediment prior to centrifugation, as this may reduce the loss of uHMW gDNA captured in the pellet.
SECTION: Sample lysis
6. Use a wide bore P1000 tip to transfer the supernatant to its corresponding 2 mL Lo-Bind tubes containing 150 μL cold potassium acetate solution (4 °C; 5 M; pH 7.4), and carefully mix until homogeneous by inversion or pipetting with the same wide bore tip.

gDNA is highly susceptible to mechanical degradation from this point onwards; handle samples with care.

The precipitate should appear whitish, opaque and slightly viscous.
SECTION: Sample lysis
7. Incubate the samples on wet ice for 5 minutes (precipitated samples can be left on wet ice for up to an hour if a break is required).

500 μL of Sera-Mag™ Speedbead solution and 175 μL of AMPure® PB beads per sample should now be removed from the fridge to equilibrate to room temperature.

A tabletop centrifuge should now be pre-chilled to 4 °C.
SECTION: Chloroform phase separation
8. Perform the first chloroform separation (C:IA) in a chemical fume hood:
SECTION: Chloroform phase separation
8.1. Add 700 μL cold chloroform:isoamyl alcohol (–20 °C; 24:1, v/v) to the samples.
SECTION: Chloroform phase separation
8.2. Mix on a tube rotator at 25 rpm for 10 minutes at room temperature.
SECTION: Chloroform phase separation
8.3. Centrifuge at 13,000 rpm for 5 minutes at 4 °C.
SECTION: Chloroform phase separation
8.4. Transfer up to 700 μL of the aqueous phase (top layer) to a fresh 2 mL Lo-Bind tube using a wide bore P1000 tip.

Carefully aspirate from the top of the aqueous phase to avoid ’dragging’ contaminants from the interphase into the pipette.
SECTION: Chloroform phase separation
9. Perform the second chloroform separation (C:IA) in a chemical fume hood:
SECTION: Chloroform phase separation
9.1. Add 700 μL cold chloroform:isoamyl alcohol (–20 °C; 24:1, v/v) to the sample.
SECTION: Chloroform phase separation
9.2. Mix on a tube rotator at 25 rpm for 10 minutes.
SECTION: Chloroform phase separation
9.3. Centrifuge at 13,000 rpm for 5 minutes at 4 °C.
SECTION: Chloroform phase separation
9.4. Transfer up to 600 μL of the aqueous phase (top layer) to the applicable empty well of the ‘Sample Plate’ (see step 12) using a wide bore P1000 tip.

Carefully aspirate from the top of the aqueous phase to avoid ’dragging’ contaminants from the interphase into the pipette.
SECTION: Chloroform phase separation
10. Add 500 µL Sera-Mag™ SpeedBead solution to each sample in the ‘POE Sample Plate’.
SECTION: Loading and Running the KingFisher™ Apex
11. Label seven KingFisher™ 1 mL 96-well deep-well plates and one KingFisher™ 200 µL standard 96-well plate with the following labels, and fill all applicable wells of each plate with their corresponding reagents (see table below).
 
   Plate namePlate TypeReagent(s) requiredPOE Tip plate 11 mL96-well tip comb (no reagent)POE Sample plate1 mLUp to 600 μL aqueous phase of sample + 500 μL Sera-Mag™ Speedbead solutionPOE Ethanol wash 1.11 mL1 mL 80% ETOHPOE Ethanol wash 1.21 mL1 mL 80% ETOHPOE Elution Plate 11 mL400 µL Buffer EBPOE Tip plate 21 mL96-well tip comb (no reagent)POE Ethanol wash 21 mL1 mL 80% ETOHPOE Elution Plate 2200 µL135 µL Buffer EB

SECTION: Loading and Running the KingFisher™ Apex
12. Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex POE Protocol Script/attached KFX file in the Materials section) and select using the play button.
SECTION: Loading and Running the KingFisher™ Apex
13. Load the filled plates onto the instrument following the instructions provided on screen and initiate once ready.
SECTION: Loading and Running the KingFisher™ Apex
14. The instrument will prompt once the inital 1X SpeedBead SPRI is finished: add 175 µL (0.45X) AMPure PB beads to each well containing sample of the ‘POE Elution Plate 1’, place the plate back into the instrument, and continue the run.
SECTION: Loading and Running the KingFisher™ Apex
15. The instrument will prompt when the 0.45X AMPure PB SPRI is finished: remove the ‘POE Elution Plate 2’ and use a wide bore p200 tip to transfer the 135 µL sample eluate to an appropriate tube for gDNA storage.
SECTION: Loading and Running the KingFisher™ Apex
16. Incubate the sample at RT overnight to allow the uHMW gDNA to solubilise.
SECTION: Loading and Running the KingFisher™ Apex
17. Proceed to appropriate QC checks and downstream processing.
SECTION: Loading and Running the KingFisher™ Apex
17.1. The gDNA extract can be stored long-term at 4 °C.