TITLE: Sanger Tree of Life HMW DNA Extraction: Automated MagAttract for Small Arthropods
AUTHORS: Amy Denton, Abitha Thomas, Caroline Howard

[DESCRIPTION]
This protocol describes the automated extraction and SPRI of HMW DNA from small arthropod samples intended for long-read sequencing using the Qiagen MagAttract HMW DNA extraction kit and the Thermo Fisher KingFisher™ Apex. This protocol is primarily effective for the class Arachnida and order Isopoda within Arthropoda covered by the Tree of Life Programme. This protocol has resulted in successful extractions for a number of species including Linyphia triangularis, Anilocra frontalis, Lekanesphaera levii, Cymodoce truncata, Porcellio scaber, Idotea baltica and Ligia oceanica, as well as a reference genome generation for Troglohyphantes excavatus.

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 primarily adapted from the 'HMW DNA Extraction of Jumping Spiders v.4' protocol; this was developed through R&D by the Meier/Jiggin group for jumping spider tissue, which uses elements of the Qiagen MagAttract protocol and the Lawniczak Lab extraction v.6 for small insects. This protocol also incorporates the pre-shear SPRI element of the 'Sanger Tree of Life HMW DNA Extraction: Automated MagAttract v.2' protocol.


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.
Remove the AMPure PB beads from the fridge 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 24 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. Prepare a lysis buffer mastermix:

 

SECTION: Sample Lysis
4. For samples which have been cryogenically homogenised via cryoPREP:
Transfer 25 mg of the sample into a 2 mL microcentrifuge tube, then hold on dry ice to keep the sample frozen.
Add 187 µL of the lysis buffer master mix to sample, then homogenise the sample and master mix by gently pipetting 10 times with a wide-bore pipette tip.
SECTION: Sample Lysis
3. For PowerMashed samples (weight less than 25 mg):
Transfer sample into a 1.5 mL BioMasher II tube and add 187 µL lysis buffer.
Disrupt sample in lysis buffer using the Diagnocine PowerMasher II tissue disruptor and BioMasher pestle, until no large pieces remain or sample cannot be disrupted further. Try to be as gentle as possible when using the PowerMasher – if samples are very small, you may wish to manually disrupt the tissue using the BioMasher pestle by hand. (For more detailed instructions regarding PowerMashing, please refer to the Sanger Tree of Life Sample Homogenisation: PowerMash protocol.)
Transfer the entire contents of the BioMasher tube to a 2 mL microcentrifuge tube using a wide-bore tip.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
6. While samples are lysing, label seven 1 mL 96-well deep-well KingFisher™ plates and two 200 µL 96-well standard plates, then fill the number of wells required for the number of samples in each plate as follows:

 

SECTION: Sample Lysis
5. Centrifuge sample tubes briefly in a mini-centrifuge and then incubate at 25 °C for 3 hours.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
7. Once samples have completed lysing, remove sample tubes from the heat block and briefly centrifuge in a mini-centrifuge to spin down.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
8. Using a wide bore pipette tip, set the volume to 200 µL, transfer lysate to the appropriate well in the sample plate taking care not to transfer large pieces of debris.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
9. 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
10. Load the filled plates onto the instrument following the instructions provided on screen.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
11. 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 66 minutes.
SECTION: Loading and Running the KingFisher™ Apex for DNA Extraction
12. 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
13. 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 DNA Extraction
14. Using a 200 µL multi-channel pipette and wide-bore tips, pipette eluates from Elution Plate 1 and Elution Plate 1 into a new 1 mL 96-well deep-well KingFisher™ plate and gently pipette mix 5–10 times with wide-bore tips to fully homogenise DNA. This new 1 mL 96-well deep-well KingFisher™ plate is now the ‘Sample Plate’ for the 0.45X SPRI.

SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
15. Set-up the KingFisher™ plates for the 0.45X SPRI as detailed below:

 

SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
16. 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.
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
17. Load the filled plates onto the instrument following the instructions provided on screen.
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
18. Once the final plate is loaded, the protocol will automatically begin; this will take approximately 40 minutes.
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
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 the 0.45X SPRI
20. Using a wide-bore pipette tip, transfer the 130 µL of eluate from the elution plate into microcentrifuge tubes. 
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
21. Incubate the DNA at room temperature overnight and perform the required QC the following morning.
SECTION: Loading and Running the KingFisher™ Apex for the 0.45X SPRI
22. Store the DNA at 4 °C.
SECTION: Sample Lysis
2. Set a heat block to 25 °C.