TITLE: Sanger Tree of Life Fragmented DNA clean up: Automated SPRI
AUTHORS: graeme oatley, Filipa Sampaio, Caroline Howard

[DESCRIPTION]
This protocol describes the automated clean up and shorter fragment removal from fragmented DNA following the Sanger Tree of Life HMW DNA Fragmentation protocols, using PacBio AMPure PB beads and the Thermo Fisher KingFisher™ Apex. This process is highly effective for sheared DNA from all of the taxonomic groups covered by the Tree of Life Programme. The output of this protocol is DNA which can be submitted for long read sequencing, including PacBio sequencing following Low Input (LI) or Ultra-Low Input (ULI) library preparation. This protocol was adapted from Sanger Tree of Life Fragmented DNA clean up: Manual SPRI to include automation for a higher throughput of samples.


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

[BEFORE_START]
AMPure PB beads are stored in the fridge at 4 °C – take them out 30 minutes before use to allow beads to equilibrate to room temperature.
Prepare fresh 80% ethanol – 80% EtOH is hygroscopic and should be prepared fresh each time to achieve optimal results using 100% absolute ethanol and nuclease free water.

[GUIDELINES]
For DNA sheared using the Sanger Tree of Life HMW DNA Fragmentation: Diagenode Megaruptor® 3 for PacBio HiFi protocol or the Sanger Tree of Life HMW DNA Fragmentation: g-Tube for ULI PacBio protocol, use a 0.6X AMPure PB beads to DNA volume.
For DNA sheared using the Sanger Tree of Life HMW DNA Fragmentation: Diagenode Megaruptor® 3 for LI PacBio protocol, use a ratio of 1X AMPure PB beads to DNA volume.
For DNA sheared using the Sanger Tree of Life HMW DNA Fragmentation: g-Tube for ULI PacBio protocol, use a ratio of 0.6X AMPure PB beads to DNA volume.
To allow for any evaporation occurring whilst on the KingFisher™ Apex, for QC to be performed and to meet internal requirements at Sanger for sequencing, 55 µL of EB is added in step 13 (3 µL is for QC and 45.4 µL for sequencing), however any volume of EB buffer can be used to elute the sheared DNA.


Additional Notes:
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.

[STEPS]
SECTION: Laboratory protocol
1. Normalise the volumes of all sheared DNA samples to the sample with the largest volume.
SECTION: Laboratory protocol
2. Set-up the KingFisher™ plates for the automated SPRI as detailed below:

   Plate namePlate typeReagent(s) requiredTip plateKingFisher‱ Apex 1 mL 96-well deep-well plateKingFisher‱ Apex 96-well tip combSample plateKingFisher‱ Apex 1 mL 96-well deep-well plateSheared DNA - variable volume+AMPure PB beads - variable volumeWash plateKingFisher‱ Apex 1 mL 96-well deep-well plate800 µL 80% ethanol (freshly made)Elution plateKingFisher‱ Apex 200 µL standard 96-well plate55 µL Buffer EB 

 
SECTION: Laboratory protocol
3. Load the normalised samples into the 1 mL 96-well deep-well plate sample plate and ensure that the 80% ethanol and the EB buffer have been loaded into the same wells within the wash plate and elution plate respectively, corresponding to that of the samples.
SECTION: Laboratory protocol
4. Vortex the now room temperature AMPure PB beads and add the amount of beads required for the desired bead:sample ratio to the sample wells in the sample plate.
SECTION: Laboratory protocol
5. Select the automated SPRI protocol on the KingFisher™ Apex (details in the KingFisher™ Apex Automated SPRI Protocol Script/attached KFX file in the Materials section).
SECTION: Laboratory protocol
6. Modify the sample plate volumes on KingFisher protocol to reflect the volumes that you have loaded by choosing the pencil icon when the protocol is highlighted; this will allow the protocol to be edited. Navigate to protocol steps and select the sample plate in the mix step. Change the bead and sample volume as required to reflect the sample:bead volume and ratio used.  
SECTION: Laboratory protocol
7. Once the sample plate volumes have been modified, use the play button to initiate the protocol and load the plates as prompted. 
SECTION: Laboratory protocol
8. Once the final plate is loaded, the protocol will automatically begin; this will take around 35 minutes to complete.
SECTION: Laboratory protocol
9. Once finished, remove the elution plate from KingFisher™ Apex and follow the on-screen instructions to remove the plates from the instrument.
SECTION: Laboratory protocol
10. Using a wide-bore pipette tip, transfer the eluates from the elution plate into 1.5 mL DNA Lo-Bind microcentrifuge tubes.
SECTION: Laboratory protocol
11. Perform QC as required.
SECTION: Laboratory protocol
12. Store samples at 4 °C.