TITLE: Sanger Tree of Life HMW DNA Extraction: Automated Nucleated Blood Nanobind®
AUTHORS: Pacific Biosciences, Adam Bates, Amy Denton, Caroline Howard

[DESCRIPTION]
This protocol describes the automated extraction of HMW DNA from nucleated blood samples intended for long-read sequencing using the Nanobind® tissue kit and the Thermo Fisher KingFisher™ Apex. It follows the "Nanobind® HMW DNA Extraction from 5 μL nucleated red blood cell on KingFisher Apex" R&D protocol, which was developed by Pacific Biosciences and validated by the Tree of Life Core Laboratory using bird, fish and reptile nucleated blood samples. This process is effective for any species with nucleated blood within the chordate group, covered by the Tree of Life Programme, however difficulties can arise with samples where preservation has not been optimal. In addition to allowing a higher throughput of nucleated blood samples, this protocol is also particularly beneficial for nucleated blood samples where clumping has occurred, with higher yields obtained compared to processing them using the Manual Nucleated Blood Nanobind® protocol. The output of this protocol is HMW DNA of high quality and quantity, which can be directed towards the HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio protocol.


Acronyms:
HMW: high-molecular weight
LI: low input

[BEFORE_START]
Add 100% ethanol to the Buffers CW1 and CW2 as per manufacturer’s instructions.
Set heat block to 37 °C (if using frozen blood) and incubate for 15 minutes to thaw the sample thoroughly.

[GUIDELINES]
This protocol follows the Nanobind® HMW DNA Extraction from 5 μL nucleated red cell blood on KingFisher Apex R&D protocol, with the inclusion of the standard sample inputs and the standard elution volumes used by Sanger Tree of Life.
This protocol is suitable for nucleated blood from birds, amphibians, reptiles or fish that has been either flash frozen or stored in ethanol.
Before starting the protocol, keep samples on dry ice to maintain temperature and prevent nucleic acid degradation.
Ensure that samples are spaced adequately from one another in the 96-well deep-well plate -  e.g. two to three empty wells between each sample - this will prevent contamination if the Nanobind disc falls from the tip comb during movement between plates.
An experienced operator can expect to comfortably process 8 to 16 samples with a start to finish period of 1 to 2 hours. This estimation excludes overnight incubation at room temperature and 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.


Troubleshooting:
Nanobind disc is not present in the Elution Plate after the completion of the protocol - The Nanobind discs occasionally remain on the tip comb after the elution and are returned to the Tip Plate at the end of the protocol. Check the Tip Plate for the Nanobind disc and if it is present, continue to QC with this sample, as this should not have any effects on the sample.
If the Nanobind disc is not in the Tip Plate, then this means that the disc became dislodged from the tip comb during the protocol and likely does not contain any DNA. For these samples, if there is material remaining, the protocol may be re-run with a new sample. Alternatively, if the Nanobind disc has been dropped in one of the wash plates, it is possible to recover the disc from this plate and continue with the protocol manually, following Sanger Tree of Life HMW DNA Extraction: Manual Nucleated Blood Nanobind.
Two Nanobind discs are present in one sample well of the Elution Plate after the completion of the protocol  - If two Nanobind discs have ended up in one sample well of the Elution Plate, then one of the discs has been dislodged from the tip comb and ended up in another sample well. This therefore means that there is contamination. If there is material remaining for the affected samples, then the protocol should be re-run with new samples. Ensure that there is sufficient distance between the sample wells to prevent this from recurring.

[STEPS]
SECTION: Laboratory Protocol
1. In one Thermo Fisher KingFisher™ 1 mL 96-well deep-well plate, labelled Lysis Plate, add the following reagents into each well for the number of samples being processed:
SECTION: Laboratory Protocol
2. Set up the six remaining Thermo Fisher KingFisher™ 1 mL 96-well deep-well plates required for the protocol as detailed in the table below. Ensure that the reagents are added into the wells corresponding to those in which samples were added into the Lysis Plate:

 

SECTION: Laboratory Protocol
3. Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details below in KingFisher™ Apex DNA Extraction Protocol section/attached file) and select using the play button.
SECTION: Laboratory Protocol
4. Load the filled plates onto the instrument following the instructions provided on screen. Once the final plate is loaded, the protocol will automatically begin; this takes approximately 75 minutes.
SECTION: Laboratory Protocol
5. Approximately 22 minutes into the protocol, the instrument will prompt the user to remove the Lysis Plate from the instrument and add 300 μL of isopropanol to each well containing a sample. 
Ensure that the isopropanol is added gently against the side of the well rather than directly into the lysis solution, as this may affect extraction purity. 
Once this has been done, re-insert the plate into the instrument and press ‘Next’ to resume the protocol.
SECTION: Laboratory Protocol
6. Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
SECTION: Laboratory Protocol
7. Using a standard P200 pipette and 200 μL pipette tips, transfer eluates from the Elution Plate to microcentrifuge tubes for storage.
SECTION: Laboratory Protocol
8. Pipette mix the eluate 10 times with a standard P200 pipette and 200 μL pipette tip to homogenise the DNA within the sample and disrupt any viscous regions.
SECTION: Laboratory Protocol
9. Allow the eluates to incubate at room temperature overnight to allow the DNA to solubilise.
SECTION: Laboratory Protocol
10. Following the overnight rest, pipette mix 10 times with a standard P200 pipette and 200 μL pipette tip, then perform the required QC.
SECTION: Laboratory Protocol
11. Store the DNA at 4 °C.
SECTION: Laboratory Protocol
1.1. Add 20 μL of Proteinase K. 
SECTION: Laboratory Protocol
1.2. Add 5 to 10 μL of nucleated blood.
SECTION: Laboratory Protocol
1.3. Add 190 to 195 μL of PBS.
SECTION: Laboratory Protocol
1.4. Add 20 μL of RNAse A.
SECTION: Laboratory Protocol
1.5. Add 150 μL of Buffer BL3. Note: add this reagent gently against the side of the well, as adding it directly into the lysis solution may affect the extraction performance.