Sanger Tree of Life Fragmented DNA clean up: Automated SPRI
Caroline Howard, graeme oatley, Filipa Sampaio
DNA clean up
solid phase reversible immobilisation
SPRI
automated SPRI
KingFisher
AMPure PB beads
reference genome
long read sequencing
Abstract
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.
Steps
Laboratory protocol
Normalise the volumes of all sheared DNA samples to the sample with the largest volume.
Set-up the KingFisher™ plates for the automated SPRI as detailed below:
| A | B | C |
|---|---|---|
| Tip plate | 1 mL 96-well deep-well plate | 96-well tip comb |
| Sample plate | 1 mL 96-well deep-well plate | Sheared DNA - variable volume + AMPure PB beads - variable volume |
| Wash plate | 1 mL 96-well deep-well plate | 800 µL 80% ethanol (freshly made) |
| Elution plate | 200 µL standard 96-well plate | 55 µL Buffer EB |
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.
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.
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).
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.
Once the sample plate volumes have been modified, use the play button to initiate the protocol and load the plates as prompted.
Once the final plate is loaded, the protocol will automatically begin; this will take around 35 minutes to complete.
Once finished, remove the elution plate from KingFisher™ Apex and follow the on-screen instructions to remove the plates from the instrument.
Using a wide-bore pipette tip, transfer the eluates from the elution plate into 1.5 mL DNA Lo-Bind microcentrifuge tubes.
Perform QC as required.
Store samples at 4 °C.
