TITLE: Sanger Tree of Life HMW DNA Fragmentation: Covaris g-TUBE for ULI PacBio
AUTHORS: graeme oatley, Filipa Sampaio, Lucy Kitchin, Raquel Juliana Vionette do Amaral, Caroline Howard

[DESCRIPTION]
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

[GUIDELINES]
The DNA sheared using this protocol must be intended for ULI PacBio sequencing.
This protocol is for the shearing of DNA into fragment sizes of 8–10 kb.
150 ng of unsheared DNA is ideal for this protocol, but 90–150 ng total input can be used.


Troubleshooting:

Low concentration samples
In some cases you may need to centrifuge >150 µL in order to process 150 ng. In this scenario, split your DNA sample into 100 µL or 150 µL subsamples, and centrifuge each subsample through the same g-TUBE. Re-combine the outputs from each centrifugation in preparation for downstream processes.

Sample does not spin through the filter
If, after three spins, the sample has not passed through the filter and still remains in the chamber, try the following:
Flip the g-TUBE (screw cap down), and spin at 4,300 x g for 30 seconds.
Recover the sample from the cap following the usual procedure (step 11).
Filter the sample with a 0.45 µm filter.
Use a new g-TUBE and follow the normal protocol. 


[STEPS]
SECTION: Laboratory protocol
1. 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.
SECTION: Laboratory protocol
2. Prior to transferring the DNA sample from its original tube, first mix the DNA sample by pipetting carefully with wide-bore pipette tip.
SECTION: Laboratory protocol
3. Transfer between 100–150 µL of the DNA sample to its corresponding labelled g-TUBE; 150 µL is the ideal volume.
SECTION: Laboratory protocol
4. 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.
SECTION: Laboratory protocol
5. Secure the g-TUBEs by tightly sealing the screw-caps, using the g-TUBE prep station provided with the g-TUBEs.
SECTION: Laboratory protocol
6. 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.
SECTION: Laboratory protocol
7. 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.
SECTION: Laboratory protocol
8. 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.
SECTION: Laboratory protocol
9. 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.
SECTION: Laboratory protocol
10. 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.
SECTION: Laboratory protocol
11. Using the g-TUBE prep station to stabilise the tube, carefully unscrew the tube from the lid, leaving the lid in the prep station.
SECTION: Laboratory protocol
12. 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.
SECTION: Laboratory protocol
13. Store the sheared DNA at 4 °C until further processing.