TITLE: Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP
AUTHORS: Kimberley J Billingsley, Ramita Dewan, Laksh Malik, Pilar Alvarez Jerez, Stith Kiley, Cornelis Blauwendraat, on behalf of the CARD Long-read Team

[DESCRIPTION]
Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP

At the NIH's Center for Alzheimer's and Related Dementias (CARD) https://card.nih.gov/research-programs/long-read-sequencing we will generate long-read sequencing data from roughly 4000 patients with Alzheimer's disease, frontotemporal dementia, Lewy body dementia, and healthy subjects. With this research, we will build a public resource consisting of long-read genome sequencing data from a large number of confirmed people with Alzheimer's disease and related dementias and healthy individuals. To generate this large-scale nanopore sequencing data we have developed a protocol for processing and long-read sequencing human frontal cortex brain tissue, targeting an N50 of  ~30kb and ~30X coverage.   

†Correspondence to: Kimberley Billingsley billingsleykj@nih.gov and Cornelis Blauwendraat cornelis.blauwendraat@nih.gov

Acknowledgements:
We would like to thank the  Nanopore team (Androo Markham &Hannah Lucio), Circulomics Inc team (Jeffrey Burke, Michelle Kim, Duncan Kilburn & Kelvin Liu) and the whole CARD long-read team listed below => UCSC: Benedict Paten, Mikhail Kolmogorov, Miten Jain, Kishwar Shafin, Trevor Pesout; NHGRI: Adam Phillippy, Arang Rhie; Baylor: Fritz Sedlazeck; JHU: Winston Timp; NINDS: Sonja Scholz; NIA: Cornelis Blauwendraat, Kimberley Billingsley, Frank Grenn, Pilar Alvarez Jerez, Bryan Traynor, Shannon Ballard, Caroline Pantazis; CZI: Paolo Carnevali.

   



[STEPS]
1. 

Part 1: Brain Tissue Cutting (~2.5 hours for 16 samples)



1.1. Add dry ice to a ice bucket

1.2. Place supplies (sterile weigh boat, razor blade, labeled empty 2mL protein LoBind microcentrifuge tubes and cooling block) in dry ice and allow to chill for ~ 5 min
1.3. Obtain tissue samples from -80 °Cfreezer and place in dry ice
1.4. Wear all necessary protective equipment (lab coat, face shield, double gloved) and complete the following steps within a chemical fume hood
1.5. Weigh labeled empty tube to tare scale, ensuring tube is centered
1.6. Working in the chemical fume hood, place the chilled weighing boat on top of the cooling tissue (use right hand to grip blade and cut tissue), use left hand just above, in between tissue and in front of hood, to shield any flying pieces)
1.7. Using the chilled razor blade, gently lift the cut tissue piece and transfer to the chilled labeled empty tube and weigh immediately, place back in dry ice immediately to avoid tissue thawing
1.8. Add or remove tissue using the method outlined above as required by input specifications ~40mg for frontal cortex, DNA recovery varies based on amount of gray matter vs. white matter)
1.9. Dispose of used weighing boat in a burn box and razor blade in the sharps waste container between each sample to prevent inter-sample contamination and keep all tissue samples on dry ice when not in use
2. Part 2: TissueRuptor Brain Tissue Disruption (~3 hours)
2.1. Place 15mL round  tubes and cold Buffer CT on ice, chill centrifuge to4 °C, and warm ThermoMixer to 55 °C

2.2. Transfer brain tissue from previous steps to 15mL round tubes (keep on ice during the entire disruption process)
2.3. Add 750 µL of cold Buffer CT

2.4.  Submerge TissueRuptor probe tip in buffer and blend at max speed for 10 s(place probe tip off to side to be cleaned later)
2.5. Transfer homogenate to a 2 mL Protein LoBind microcentrifuge tube including all undisrupted tissue chunks and any foam that forms
2.6. Pellet homogenate by centrifuging at 6,000 x g and 4 °C for 5 min. Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)

2.7. Add 1 mLof cold Buffer CT and pipette mix 10X with a wide bore P1000 pipette to resuspend tissue

2.8. Pellet homogenate by centrifuging at 6,000 x g and 4 °C for 2 min. Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)
2.9. Pulse vortex for 1 sx 5 times (max setting) to dislodge pellet
2.10. Add 20 µL of Proteinase K to the previous pellet
2.11. Add 50 µL1X TE pH 8
2.12. Add 60 µL Buffer CS
2.13. Add 100 µL Buffer CLE3 and pipette mix 15X with a wide bore P200 pipette
2.14. Incubate for 60 min on a ThermoMixer at 55 °Cand 900 rpm
2.15. Spin on a mini-centrifuge for 2 s  to remove liquid from the cap
2.16. Add 20 µL of RNaseA and pipette mix 3X with a wide bore P200 pipette
2.17. Incubate for 30 minon a ThermoMixer at 55 °C and 900 rpm
2.18.  Spin the tube on a mini-centrifuge for2 s to remove liquid from the cap
2.19. Add 50 µL Buffer SB and vortex for10 s at maximum speed (transfer any foam that appears)
3. Part 3: KingFisher Apex Nanobind Tissue Big DNA protocol (~2 hours)
3.1. Prepare KingFisher Apex plates as follows:

Plate 1 - Lysis Binding: Sample + 50 µL BL3
Plate 2 - Nanobind Storage: one 3mm Nanobind disk
Plate 3 - CW1 Wash 1: 600 µLBuffer CW1
Plate 4 - CW1 Wash 2: 600 µL Buffer CW1
Plate 5 - CW2 Wash 1: 600 µL Buffer CW2
Plate 6 - CW2 Wash 2: 600 µL Buffer CW2
Plate 7 - Elution: 100 µL Buffer EB
Plate 8 - Tip: KingFisher Flex 96-Tip Combo

3.2. Run KingFisher Apex program “210804_nanobind_tissue_kf_apex_v2.kfx” (KF script available by request from Circulomics Inc)
3.3. After 12 min when the program pauses, add 300 µLIPA

3.4. Collect DNA by transferring eluate to a new 1.5 mL microcentrifuge tube OR if sample is to be sheared, the sample can be transferred to a DNA Fluid+ tube.
3.5. Let the sample rest at room temperature overnight to allow DNA to solubilize

5. Part 5: DNA Shearing (8 hours per 48 samples)


5.1.  Megaruptor 3 Shear with DNAFluid+  kit:
Normalize samples to 40-60 ng/uL in  150 µL total volume (to be made up with nuclease-free water and equates to 4000 - 6000ng per sample) in DNA Fluid+ tubes.
Attach the DNA Fluid+ needle onto the tube and push the entire item into the Megaruptor 3 slots until it fits snugly. If running fewer than 8 samples, put the tubes in the 1st and/or 8th slots, working your way in. Samples should always be balanced, if running an odd number of samples, samples can be balanced with an empty corresponding tube. Shear at speed 45 (takes around ~60 min)
Once the MR3 shearing is finished, repeat the run by navigating back to the main menu and select speed 45 again (takes around ~60 min)
Avoid any vortexing of DNA from this point on to avoid any unnecessary further shearing, instead mix by gently flicking the tube and spin down
6. Part 6: Post-shear DNA Quantification

6.1. Quantify the samples with the nanodrop and Qubit and size using the Agilent Tapestation 4200
6.2. Upload the tapestation reports and  and quantifications 
6.3. For Step 6 the starting material must be > 3ug. If a sample does not reach this requirement then repeat DNA extraction from Part 1. 
6.4. DNA can be stored at 4° C for up to four weeks, or -80 °Cindefinitely
7. 
Part 7: Library Prep (~6 hours, not including flushing and returning cells)





7.1. A. DNA Repair and End-Prep
Put all the necessary reagents on ice to thaw and the Agencourt AMPure XP beads out at room temperature
Prepare the following in a 0.2 mL thin-walled PCR tube:
    - 48 µL DNA (load 3 ug, this will likely be over 48 μL but that is fine, just adjust the amount of beads to match the total volume of this mixture)
    -3.5 µL NEBNext FFPE DNA Repair Buffer (vortex)
   - 3.5 µL Ultra II End-prep reaction buffer (vortex)
    - 3 µL Ultra II End-prep enzyme mix (do not vortex)
    - 2 µLNEBNext FFPE DNA Repair Mix (do not vortex)
3. Mix thoroughly by gently flicking tube or pipetting up and down 10X, and then spin down
4. Using a thermal cycler, incubate samples at 20 °C for5 min and 65 °C
for 5 min
- Start and pause thermal cycler to allow lid to come to 85 °Cbefore putting samples in
5. Allow Thermocycler to cool to 4 °C and then remove your samples.
6. Resuspend the AMPure XP beads by vortexing
7. Transfer DNA samples to clean 1.5 mL Eppendorf DNA LoBind tube
8. Add60 µL(or equivalent volume, see step 2) of resuspended beads to the reaction and mix by pipetting up and down 10X
9. Incubate for 5 min at room temperature
10. Prepare 500 µL per sample of fresh 75% ethanol in Nuclease-free water
11. Pellet sample on magnet until eluate is clear and colorless, about 2 min

12. Pipette off the supernatant and retain, just in case the following quant is uncharacteristically or surprisingly low
13. With the samples remaining on the magnet, wash the beads with200 µL of the ethanol, pipetting on the opposite wall (the goal here is to make sure the beads are fully covered) and making sure not to disturb the pellet, count to 3 and remove and discard ethanol
14. Repeat previous step
15. Spin down and place the tube back on magnet, pipetting off any residual ethanol
16. Allow to dry for ~30 s but do not over-dry
17. Remove the tube from the magnetic rack and resuspend the pellet in 61 µLNuclease-free water, incubate for 3 min at room temperature (hold in hands and gently flick every so often)
18. Pellet the samples on a magnet until eluate is clear and colorless
19. Remove and retain61 µL  of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube
20. The sample concentration must be > 40ng/ul. If the sample does not reach this requirement restart from Part 6. 
21. It is possible to store samples at4 °C overnight at this step if needed

7.2. B. Adapter Ligation and Clean-Up
Spin down the AMX-F, Quick T4 ligase, and LNB, then return to ice
- Do not allow AMX-F to remain at room temperature for too long
2. Thaw LNB at RT and mix by pipetting up and down (vortexing is ineffective due to viscosity)
3. Thaw EB at RT, mix by vortexing, spin down, and place on ice
4. Thaw SFB at RT, mix by vortexing, spin down, and keep at RT
5. In a 1.5 mL Eppendorf DNA LoBind tube, mix the following in order:
    - 60 µLDNA sample (if not 60, add water until it is)
    - 25 µL LNB
    - 10 µL Quick T4
    -5 µLAMX-F
6. Mix gently by flicking the tube and spin down
7. Incubate the reaction for 10 minat RT
8. During this time, put flow cells out at RT
9. Resuspend beads by vortexing
10. Add 45 µL of resuspended beads to the reaction and mix by flicking
11. Incubate for 5 minat RT
12. Spin down sample and pellet on magnet
13. Pipette off the supernatant and retain, just in case the final elution quant is uncharacteristically or surprisingly low
14. Wash the beads with 250 µL SFB, flick to resuspend and repellent, remove and discard supernatant
15. Repeat previous step
16. Spin down and place the tube back on magnet, pipetting off any residual SFB
17. Allow to dry for ~30 s, but do not over-dry
18. Remove the tube from magnet and resuspend pellet in 25 μL EB, spin down, and incubate for 20 min at 37 °C
19. During this time, QC the flow cells (only use flow cells with >6000 pores)
20. Pellet the beads on magnet until eluate is clear and colorless
21. Remove and retain 25 µL of eluate into a clean 1.5 mL Eppendorf DNA LoBind tube (this is the DNA library)
22. Quantify samples on Qubit
23. Reprep library  from Part  6 if < 1200 ng. 
24. Keep libraries on ice until ready to load on flow cell
7.3. C. Priming and Loading Flow Cell
Thaw SBII, FLT, and FB, vortex, and spin down
Thaw LBII
Add 30 µL of thawed and mixed FLT directly to tube of FB and vortex
Expose inlet port on flow cell and draw back a small volume to remove any air bubbles (usually about 20-30 μL, just until a small volume of buffer enters the pipette tip)
Flush 500 µL of Priming Mix into the inlet port of the flow cell, being extremely careful to avoid the introduction of air bubbles at the end
Wait 5 min
During this time, separate the DNA library into three equal aliquots (ideally with 400 ng of DNA each). Bring each aliquot up to 24 µL with EB. (i.e, if your final elution is exactly 1200 ng in 24 ul, move 8 µLto three separate tubes and add 16 µLof EB to each.)
Prepare the first library  mix for loading:
    -  75 µL SBII (vortex)
    - 51 µL LB (pipette up and down immediately before use)
     - 24 µLDNA library in EB (400 ng) 
9. Immediately load all 150 µLof the library mix
10. Close valve to seal inlet port and close PromethION lid
11. Wait 10 minand then initiate sequencing
12. Ideally, the library quants yielded at least 1200 ng to allow for 3X 400 ng loads, the latter 2 loaded approximately after 24 and 48 hours. However this will vary slightly depending on pore usage, data generated, as well as other factors i.e. if after 24 hours there are still +3000 pores then the sample does not need to be reloaded until 48 hours.
        - To wash and reload a flow cell, begin by thawing Wash Mix (WMX) on ice and Wash Diluent (DIL) at RT (DIL should be vortexed, WMX should NOT be vortexed, only spun)
        - Add2 µLWMX into 398 µLDIL and pipette mix
        -Pause the PromethION runs and export .pdf reports
        -Rotate the inlet port cover to reveal inlet port 1
        -Using a P1000, insert tip into inlet port and draw back a small volume using the wheel to remove any air (usually around 20-30 μL)
         -Load 400 µLFlow Cell Wash Mix into the inlet port, avoiding any introduction of air
         -Wait 60 min
        -Repeat priming steps and reload samples (steps 1 - 12)






7.4. D. Flushing and Recycling Flow Cells (~15 minutes per set of 4 flow cells)
Following the completion of the sequencing, flow cells may be removed from the sequencer
Place enough absorbent material to take up approximately 4 mL of flush waste
Rotate valve to reveal inlet port 1
Place flow cell at a 45° angle on the absorbent material and, using a P1000, flush 1 mL of DI water into the inlet port
Repeat 3 more times for a total of 4 mL
Once complete, close the input port cover and remove all liquid from the waste port
Dispose of absorbent material as local biological waste guidelines dictate
 8. Return flow cells to clear plastic tray in which it was shipped, making sure to record the flow cell IDs
9. Seal the tray with the sticker provided in the packaging
10. Put the clear plastic lid back on the tray
11. Place the tray back in the packaging
12. Place packaged cells in the returns box (large box can hold 80)
13. Once returns box is filled, follow the instructions here and follow the prompts to request the box to be sent back to Nanopore


SECTION: Sequencing results:
8. 

Following 72 hours of sequencing the sample should yield an N50 ≥ 30kb with a data output ~ 100-160GB. 
SECTION: Sequencing results:
9.    
  

4. Part 4. Pre-Shear DNA Quantification 
4.1. Hand shear DNA 
Hand-shear 10X with 1mL Luer-Lock syringes and 1.5” needles (bringing sample up into needle and depressing plunger counts as 1 cycle)
4.2. Quantify
Quantify the samples with the nanodrop and Qubit and size using the Agilent Tapestation 4200
Upload the tapestation reports
Add the quantifications
Only take forward samples that  are > 3ug. If a sample does not reach this requirement then repeat DNA extraction from Part 1.