Sanger Tree of Life HMW DNA Fragmentation: Covaris g-TUBE for ULI PacBio
Caroline Howard, graeme oatley, Raquel Juliana Vionette do Amaral, Filipa Sampaio, Lucy Kitchin
Abstract
This protocol describes the centrifugation-mediated fragmentation of HMW DNA from samples prepared via any of the Sanger Tree of Life HMW DNA extraction protocols. The protocol produces fragments in the 8–10 kb size range using the Covaris g-Tube. The sheared DNA is suitable for downstream long read sequencing, including PacBio sequencing after ultra-low input (ULI) library preparation. This process is highly effective for DNA extracts across all taxonomic groups in the Tree of Life Programme. The output of this protocol is sheared DNA, which can be directed towards fragmented DNA clean up, using either the Manual or Automated SPRI protocols.
Acronyms and abbreviations
HMW: high molecular weight
SPRI: solid-phase reversible immobilisation
ULI: ultra-low input
Steps
Laboratory protocol
Label the required number of Covaris g-TUBEs for each DNA sample that will be sheared; ensure that the tubes are labelled both on the lid and on the bottom.
Prior to transferring the DNA sample from its original tube, first mix the DNA sample by pipetting carefully with wide-bore pipette tip.
Transfer between 100–150 µL of the DNA sample to its corresponding labelled g-TUBE; 150 µL is the ideal volume.
If processing multiple samples, ensure that the volumes are equal by adding EB buffer to normalise the volumes. Be aware that the processing time from transfer of the sample into the g-TUBE and the g-TUBE containing the sample into the centrifuge should be no longer than 15 minutes, to avoid the sample migrating into the filter whilst on the bench.
Secure the g-TUBEs by tightly sealing the screw-caps, using the g-TUBE prep station provided with the g-TUBEs.
Place the g-TUBEs into a bench-top centrifuge. If there is an uneven number of samples, make sure to balance the centrifuge using a spare g-TUBE.
Run for 1 minute at 5,000 rpm for a shear size of 10 kb; refer to the manufacturer's instructions for conditions to obtain different shear sizes.
Repeat the centrifugation in step 7 until all the volume has passed through the filter, up to three times. Check the tube between chambers to ensure that all of your sample has passed through the filter. If the sample has not passed through the filter after the third spin, please refer to the ‘Troubleshooting’ section in the Guidelines section.
Invert the g-TUBEs within the prep station so the fixed base is facing up and the screw-cap is facing down, then repeat steps 6, 7 and 8 so that all of the DNA has passed completely through the g-TUBE filter a total of two times.
Once all of the sample has passed through the filter, remove the g-TUBEs from the centrifuge whilst maintaining their current orientation (screw-cap facing down), and place them screw-cap side down into the g-TUBE prep station.
Using the g-TUBE prep station to stabilise the tube, carefully unscrew the tube from the lid, leaving the lid in the prep station.
Aspirate the sample from the screw-cap using a standard pipette tip and transfer the sheared DNA to a fresh DNA Lo-Bind microcentrifuge tube.
Store the sheared DNA at 4 °C until further processing.
