Jun 22, 2023

Public workspaceProcessing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP

DOI
dx.doi.org/10.17504/protocols.io.kxygx3mqkg8j/v1
  • Kimberley J Billingsley1,2,
  • Ramita Dewan2,
  • Laksh Malik1,
  • Pilar Alvarez Jerez1,2,
  • Stith Kiley1,
  • Cornelis Blauwendraat1,2,
  • on behalf of the CARD Long-read Team1
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging, Bethesda, Maryland, USA;
  • 2Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA
Kimberley J Billingsley
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DOI: dx.doi.org/10.17504/protocols.io.kxygx3mqkg8j/v1
Protocol Citation: Kimberley J Billingsley, Ramita Dewan, Laksh Malik, Pilar Alvarez Jerez, Stith Kiley, Cornelis Blauwendraat, on behalf of the CARD Long-read Team 2023. Processing human frontal cortex brain tissue for population-scale Oxford Nanopore long-read DNA sequencing SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3mqkg8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 22, 2023
Last Modified: August 08, 2023
Protocol Integer ID: 83880
Disclaimer
In development
We are still developing and optimizing this protocol.
Abstract
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.

Figure 1. Overview of HMW Brain DNA extraction and ONT sequencing protocol 



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Materials
List of reagents/equipment needed:
Consumables:
DescriptionSupplier
1.5mL DNA LoBind tubesQiagen
1.5mL Protein LoBind tubesQiagen
Sterile Weigh Boat
Razor Blade
Cooling Block
15mL conical-bottom tube
TissueRuptor Disposable Tips
2mL Protein LoBind tubes
3mm Nanobind DisksCirculomics -Tissue big DNA kit
1mL Luer-lock Syringes
1.5” Needles
DNA Fluid+ KitDiagenode
0.2mL thin-wall PCR tubes
PromethION Flow CellsOxford Nanopore Technologies
Reagents:


DescriptionSupplier
Nanobind Tissue Big DNA KitCirculomics
TE pH 8Millipore Sigma
Isopropyl Alcohol
Agencourt AMPure XP beadsAgencourt 
NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation SequencingNew England BioLabs
Ligation Sequencing Kit (SQK-LSK110)Oxford Nanopore Technologies
Flow Cell Wash Kit (EXP-WSH004)Oxford Nanopore Technologies
Ultra Pure WaterKD Medical
1x dsDNA BR Working SolutionThermo Fisher
Absolute Ethanol
Genomic DNA Screen TapeAgilent
Equipment:

DescriptionSupplier
Chemical Fume Hood
Weigh Scale
Centrifuge 5425REppendorf
ThermoMixer CEppendorf
TissueRuptor IIQiagen
KingFisher ApexThermo Fisher
MegaruptorDiagenode
C1000 Touch ThermocyclerBioRad
NanoDrop 8000Thermo Fisher
Qubit 4Thermo Fisher
Tapestation 4200Agilent
Vortex Genie-TScientific Industries
Microfuge
Magnetic separator (suitable for 1.5mL Eppendorf tubes)
PromethION 48Oxford Nanopore Technologies


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


Add dry ice to a ice bucket

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 ~ Duration00:05:00
5m
Obtain tissue samples from Temperature-80 °C freezer and place in dry ice
Wear all necessary protective equipment (lab coat, face shield, double gloved) and complete the following steps within a chemical fume hood
Weigh labeled empty tube to tare scale, ensuring tube is centered
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)
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
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)
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
Part 2: TissueRuptor Brain Tissue Disruption (~3 hours)
Place 15mL round tubes and cold Buffer CT on ice, chill centrifuge toTemperature4 °C , and warm ThermoMixer to Temperature55 °C

Transfer brain tissue from previous steps to 15mL round tubes (keep on ice during the entire disruption process)
Add Amount750 µL of cold Buffer CT

Submerge TissueRuptor probe tip in buffer and blend at max speed for Duration00:00:10 (place probe tip off to side to be cleaned later)
10s
Transfer homogenate to a 2 mL Protein LoBind microcentrifuge tube including all undisrupted tissue chunks and any foam that forms
Pellet homogenate by centrifuging at 6,000 x g and Temperature4 °C for Duration00:05:00 . Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)

5m
Add Amount1 mL of cold Buffer CT and pipette mix 10X with a wide bore P1000 pipette to resuspend tissue

Pellet homogenate by centrifuging at 6,000 x g and Temperature4 °C for Duration00:02:00 . Discard supernatant (pellet may not be visible, so pipette carefully and avoid pipetting from the bottom of tube)
2m
Pulse vortex for Duration00:00:01 x 5 times (max setting) to dislodge pellet
1s
Add Amount20 µL of Proteinase K to the previous pellet
Add Amount50 µL 1X TE pH 8
Add Amount60 µL Buffer CS
Add Amount100 µL Buffer CLE3 and pipette mix 15X with a wide bore P200 pipette
Incubate for Duration01:00:00 on a ThermoMixer at Temperature55 °C and 900 rpm
1h
Spin on a mini-centrifuge for Duration00:00:02   to remove liquid from the cap
2s
Add Amount20 µL of RNaseA and pipette mix 3X with a wide bore P200 pipette
Incubate for Duration00:30:00 on a ThermoMixer at Temperature55 °C and 900 rpm
30m
Spin the tube on a mini-centrifuge forDuration00:00:02 to remove liquid from the cap
2s
Add Amount50 µL Buffer SB and vortex forDuration00:00:10 at maximum speed (transfer any foam that appears)
10s
Part 3: KingFisher Apex Nanobind Tissue Big DNA protocol (~2 hours)
Prepare KingFisher Apex plates as follows:

Plate 1 - Lysis Binding: Sample + Amount50 µL BL3
Plate 2 - Nanobind Storage: one 3mm Nanobind disk
Plate 3 - CW1 Wash 1: Amount600 µL Buffer CW1
Plate 4 - CW1 Wash 2: Amount600 µL Buffer CW1
Plate 5 - CW2 Wash 1: Amount600 µL Buffer CW2
Plate 6 - CW2 Wash 2: Amount600 µL Buffer CW2
Plate 7 - Elution: Amount100 µL Buffer EB
Plate 8 - Tip: KingFisher Flex 96-Tip Combo

Run KingFisher Apex program “210804_nanobind_tissue_kf_apex_v2.kfx” (KF script available by request from Circulomics Inc)
After Duration00:12:00 when the program pauses, add Amount300 µL IPA

12m
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.
Let the sample rest at room temperature overnight to allow DNA to solubilize

Part 4. Pre-Shear DNA Quantification
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)
Quantify
  1. Quantify the samples with the nanodrop and Qubit and size using the Agilent Tapestation 4200
  2. Upload the tapestation reports
  3. Add the quantifications
  4. Only take forward samples that are > 3ug. If a sample does not reach this requirement then repeat DNA extraction from Part 1.
Part 5: DNA Shearing (8 hours per 48 samples)


Megaruptor 3 Shear with DNAFluid+  kit:
  1. Normalize samples to 40-60 ng/uL in Amount150 µL total volume (to be made up with nuclease-free water and equates to 4000 - 6000ng per sample) in DNA Fluid+ tubes.
  2. 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 ~Duration01:00:00 )
  3. Once the MR3 shearing is finished, repeat the run by navigating back to the main menu and select speed 45 again (takes around ~Duration01:00:00 )
  4. 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
2h
Part 6: Post-shear DNA Quantification

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




A. DNA Repair and End-Prep
  1. Put all the necessary reagents on ice to thaw and the Agencourt AMPure XP beads out at room temperature
  2. Prepare the following in a 0.2 mL thin-walled PCR tube:
- Amount48 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)
-Amount3.5 µL NEBNext FFPE DNA Repair Buffer (vortex)
- Amount3.5 µL Ultra II End-prep reaction buffer (vortex)
- Amount3 µL Ultra II End-prep enzyme mix (do not vortex)
- Amount2 µL NEBNext 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 Temperature20 °C forDuration00:05:00 and Temperature65 °C for Duration00:05:00 - Start and pause thermal cycler to allow lid to come to Temperature85 °C before putting samples in
5. Allow Thermocycler to cool to Temperature4 °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. AddAmount60 µL (or equivalent volume, see step 2) of resuspended beads to the reaction and mix by pipetting up and down 10X
9. Incubate for Duration00:05:00 at room temperature
10. Prepare Amount500 µL per sample of fresh 75% ethanol in Nuclease-free water
11. Pellet sample on magnet until eluate is clear and colorless, about Duration00:02:00
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 withAmount200 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 ~Duration00:00:30 but do not over-dry
17. Remove the tube from the magnetic rack and resuspend the pellet in Amount61 µL Nuclease-free water, incubate for Duration00:03:00 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 retainAmount61 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 atTemperature4 °C overnight at this step if needed

20m 30s
B. Adapter Ligation and Clean-Up
  1. 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:
- Amount60 µL DNA sample (if not 60, add water until it is)
- Amount25 µL LNB
- Amount10 µL Quick T4
-Amount5 µL AMX-F
6. Mix gently by flicking the tube and spin down
7. Incubate the reaction for Duration00:10:00 at RT
8. During this time, put flow cells out at RT
9. Resuspend beads by vortexing
10. Add Amount45 µL of resuspended beads to the reaction and mix by flicking
11. Incubate for Duration00:05:00 at 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 Amount250 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 ~Duration00:00:30 , but do not over-dry
18. Remove the tube from magnet and resuspend pellet in 25 μL EB, spin down, and incubate for Duration00:20:00 at Temperature37 °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 Amount25 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
35m 30s
C. Priming and Loading Flow Cell
  1. Thaw SBII, FLT, and FB, vortex, and spin down
  2. Thaw LBII
  3. Add Amount30 µL of thawed and mixed FLT directly to tube of FB and vortex
  4. 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)
  5. Flush Amount500 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
  6. Wait Duration00:05:00
  7. During this time, separate the DNA library into three equal aliquots (ideally with 400 ng of DNA each). Bring each aliquot up to Amount24 µL with EB. (i.e, if your final elution is exactly 1200 ng in 24 ul, move Amount8 µL to three separate tubes and add Amount16 µL of EB to each.)
  8. Prepare the first library  mix for loading:
- Amount75 µL SBII (vortex)
- Amount51 µL LB (pipette up and down immediately before use)
- Amount24 µL DNA library in EB (400 ng)
9. Immediately load all Amount150 µL of the library mix
10. Close valve to seal inlet port and close PromethION lid
11. Wait Duration00:10:00 and 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)
- AddAmount2 µL WMX into Amount398 µL DIL 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 Amount400 µL Flow Cell Wash Mix into the inlet port, avoiding any introduction of air
-Wait Duration01:00:00
-Repeat priming steps and reload samples (steps 1 - 12)






1h 15m
D. Flushing and Recycling Flow Cells (~15 minutes per set of 4 flow cells)
  1. Following the completion of the sequencing, flow cells may be removed from the sequencer
  2. Place enough absorbent material to take up approximately 4 mL of flush waste
  3. Rotate valve to reveal inlet port 1
  4. Place flow cell at a 45° angle on the absorbent material and, using a P1000, flush Amount1 mL of DI water into the inlet port
  5. Repeat 3 more times for a total of Amount4 mL
  6. Once complete, close the input port cover and remove all liquid from the waste port
  7. 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


Sequencing results:
Sequencing results:


  1. Following 72 hours of sequencing the sample should yield an N50 ≥ 30kb with a data output ~ 100-160GB. 
Figure 2. Expected Read Length Histogram:
Figure 3.  Expected Muxscan (from 2 loads):