Apr 07, 2025

Public workspaceProcessing frozen human blood for population-scale SQK-LSK114 Oxford Nanopore long-read DNA sequencing SOP V.2

Version 1 is forked from Processing frozen human blood samples for population-scale  SQK-LSK114 Oxford Nanopore long-read DNA sequencing SOP
DOI
dx.doi.org/10.17504/protocols.io.5qpvo3639v4o/v2
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging, Bethesda, Maryland, USA
  • NIH Center for Alzheimer's and Related Dementias
Abigail Miano-Burkhardt
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo3639v4o/v2
Protocol Citation: Jackson Mingle, Kimberly Paquette, Abigail Miano-Burkhardt, Pilar Alvarez Jerez, Laksh Malik, Cornelis Blauwendraat, Kimberley J Billingsley, on behalf of the CARD Long-read Team 2025. Processing frozen human blood for population-scale SQK-LSK114 Oxford Nanopore long-read DNA sequencing SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3639v4o/v2Version created by Jackson Mingle
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: In development
We are still developing and optimizing this protocol
Created: January 24, 2024
Last Modified: April 08, 2025
Protocol Integer ID: 124147
Keywords: Long-read sequencing, Oxford Nanopore sequencing, High molecular weight DNA extraction, DNA extraction, Human blood extraction, Whole blood extraction, DNA size selection, DNA shearing
Disclaimer
Still in development.
Abstract
At the NIH 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 thousands of patients with Alzheimer's disease, frontotemporal dementia, and Lewy body dementia, as well as healthy subjects. With this research, we will build a public resource consisting of long-read genome sequencing data from a large number of confirmed patients 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 of frozen human blood samples, targeting an N50 of ~35-40 kb and ~30X coverage.

Acknowledgements:
We would like to thank the Nanopore team (Jade Bartolo, Olivor Holman, Androo Markham, and Jessica Anderson), PacBio team (Jeffrey Burke, Michelle Kim, Duncan Kilburn, and Kelvin Liu), and the whole CARD long-read team. Workflow graphic by Paige Jarreau.

This protocol was optimized using frozen blood samples from the PPMI initiative. PPMI – a public-private partnership – is funded by the Michael J. Fox Foundation for Parkinson’s Research and funding partners, a full list of the PPMI funding partners can be found at www.ppmi-info.org/fundingpartners.

†Correspondence to: Kimberley Billingsley billingsleykj@nih.gov

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

Materials
Materials:
AB
MaterialVendor (Part Number)
Nanobind HT 1 mL blood kitPacBio (102-762-800)
KingFisher 24 deep-well plates, barcodedThermo Fisher Scientific (95040470B)
KingFisher Apex 24 deep-well tip comb & plates, barcodedThermo Fisher Scientific (97002610B)
Ethanol (96-100%)Any major lab supplier (MLS)
Isopropanol (100%)Any MLS
1.5 mL DNA LoBind tubesEppendorf (022431021)
Qubit 1X dsDNA BR assay kitThermo Fisher Scientific (Q33266)
Qubit Flex Assay Tube StripsThermo Fisher Scientific (Q33252)
Femto Pulse gDNA 165kb analysis kitAgilent Technologies, Inc. (FP-1002-0275)
Megaruptor 3 shearing kitDiagenode (E07010003)
SRE kitPacBio (102-208-300)
Ligation Sequencing Kit XL V14Oxford Nanopore Technologies (SQK-LSK114-XL)
NEBNext Companion Module for Oxford Nanopore Technologies Ligation SequencingNew England Biolabs (E7180L)
Nuclease-free waterAny MLS
0.2 mL thin-walled PCR tubesThermo Fisher Scientific (AB-0620B)
AMPure XP reagentBeckman Coulter (A63882)
Qubit 1X dsDNA HS assay kitThermo Fisher Scientific (Q33231)
PromethION flow cellOxford Nanopore Technologies (FLO-PRO114M)
Flow Cell Wash KitOxford Nanopore Technologies (EXP-WSH004-XL)
Table 1. Required materials

Equipment:
AB
EquipmentVendor (Part Number)
KingFisher Apex systemThermo Fisher Scientific (5400940)
KingFisher Apex 24 Combi magnet headThermo Fisher Scientific (24079940)
KingFisher Apex 24 deep-well heating blockThermo Fisher Scientific (24075940)
Qubit Flex fluorometerThermo Fisher Scientific (Q33327)
Vortex mixerAny major lab supplier (MLS)
MinicentrifugeAny MLS
Femto Pulse systemAgilent Technologies, Inc (M5330AA)
Megaruptor 3 systemDiagenode (B06010003)
ThermoMixerEppendorf (5382000023)
MicrocentrifugeEppendorf (5404000413)
Thermal cyclerBio-Rad (1851197)
Platform rockerAny MLS
DynaMag-2 MagnetThermo Fisher Scientific (12321D)
PromethION 24 or 48 sequencing unitOxford Nanopore Technologies (PRO-SEQ024 or PRO-SEQ-048)
Table 2. Required equipment

Materials for NGS STAR:
AB
MaterialVendor (Part Number)
2 mL tubesSarstedt (72.693.005)
PCR ComfortLidHamilton (814300)
MIDI plateThermo Fisher Scientific (AB-0859)
HSP plateBio-Rad (HSP9601)
50 µL tipsHamilton (235979)
300 µL tipsHamilton (235903)
1000 µL tipsHamilton (235940)
60 mL reservoirHamilton (56694-01)
20 mL reservoirRoche (3004058001)
Table 3. Additional materials required for automated library preparation on Hamilton NGS STAR

Equipment for NGS STAR:
AB
EquipmentVendor (Part Number)
NGS STARHamilton (STAR)
Magnetic Stand-96Thermo Fisher Scientific (AM10027)
Table 4. Additional equipment required for automated library preparation on Hamilton NGS STAR

Part 1: Extracting HMW DNA using the Nanobind HT 1 mL blood kit for mammalian whole blood on the KingFisher Apex system
Part 1: Extracting HMW DNA using the Nanobind HT 1 mL blood kit for mammalian whole blood on the KingFisher Apex system
Obtain blood samples from -80 °C freezer. Thaw at Temperature37 °C for Duration00:15:00  in a water bath or dry block heater. Inversion mix 15-20x to thoroughly mix immediately prior to use.

Note:  The protocol requires Amount1 mL  of whole blood. If samples have a volume larger than Amount1 mL  , aliquot Amount1 mL  into 1.5mL Eppendorf DNA LoBind tubes and keep frozen at Temperature-80 °C  until ready to use. It is important to limit the amount of freeze/thaw cycles as much as possible.
Prepare the KingFisher Apex 24 deep-well plates:
  • Plate 1: Lysis/Binding Plate: Sample and reagents from step 3
  • Plate 2: Nanobind Storage Plate: One 5 mm Nanobind disk per well
  • Plate 3: Wash Plate 1: Amount2 mL Buffer CW1 per well
  • Plate 4: Wash Plate 2: Amount2 mL  Buffer CW2 per well
  • Plate 5: Wash Plate 3: Amount2 mL  Buffer CW2 per well
  • Plate 6: Elution Plate : Amount200 µL Buffer EB per well
  • Plate 7: Tip Plate: KingFisher Apex 24 deep-well tip comb

Note: Nanobind disks do not need to be perfectly centered in the wells, but ensure they are at the bottom of the wells and not stuck to the side walls.

Note: Buffer CW1 and CW2 are supplied as concentrates. This kit uses CW1 with a 60% final ethanol concentration and CW2 with a 60% final ethanol concentration. Before using, add the appropriate amount of ethanol (96–100%) to Buffer CW1 and Buffer CW2 as indicated on the bottles.
Prepare the Lysis/Binding Plate:
  • Add Amount100 µL  Proteinase K per well
  • Add Amount1 mL  whole blood per well (inversion mix 15-20x immediately prior to use)
  • Add Amount100 µL  RNase A per well
  • AddAmount750 µL  Buffer BL3 per well

Note: Add Buffer BL3 gently against the side of the well into the Lysis/Binding solution. Adding Buffer BL3 directly to the Lysis/Binding solution may affect extraction performance.

Note: The sample and reagents MUST be added to the wells in the order described above.
Ensure the KingFisher Apex instrument is set up with the 24 Combi magnetic head and the 24 deep-well heating block. 
Select the 1mL_Blood_Nanobind_HT_APEX script on the KingFisher Apex instrument. Insert plates into the KingFisher Apex instrument as indicated on the display and press ‘Next’ after every plate to confirm position. The protocol will start when the final plate is loaded and the ‘Next’ button is pressed.
When prompted by the instrument (~50 minutes after start), remove the Lysis/Binding Plate from the instrument and add Amount1.5 mL  of isopropanol to each well. Re-insert the plate and press ‘Next’ to resume the protocol.

Note: Add isopropanol gently against the side of the well into the Lysis/Binding solution. Adding isopropanol directly to the Lysis/Binding solution may affect extraction purity.
When prompted by the instrument, remove the Elution Plate from the instrument. Transfer eluate from each well to a new 1.5 mL Eppendorf DNA LoBind tube.

Note: Residual sample volume may be present in the tip comb plate. Transfer residual eluate to the 1.5 mL Eppendorf DNA LoBind tube.
Pipette-mix the sample 10x with a standard P200 pipette to homogenize and disrupt any unsolubilized “jellies” that may be present.
Let eluate rest overnight at room temperature to allow DNA to solubilize.
Following overnight rest, pipette-mix 10x with a standard P200 pipette.
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA BR Assay. Use Amount2 µL of DNA per measurement.

Note: Triplicate measurements (top, middle, and bottom) are advised for new cohorts. Additional hand-shearing with a syringe may be necessary for samples with exceptionally variable measurements.
Optional: Quantify by taking a single measurement on the NanoDrop 8000 spectrophotometer. Use Amount2 µL  of DNA per measurement.

Note: Measurement on NanoDrop 8000 is advised for new cohorts or brain regions. Additional hand-shearing with a syringe may be necessary for samples with exceptionally variable measurements.
Optional: Size on the Agilent Femto Pulse System with the Genomic DNA 165 kb kit. The expected size range for samples post-extraction is >100kb. 
Figure 2. Femto Pulse trace post-extraction

Note: Sizing on Agilent Femto Pulse System is advised for new cohorts. Additional hand-shearing with a syringe may be necessary for samples with exceptionally variable measurements.
Store at Temperature4 °C until ready for shearing. At least Amount6 µg  DNA is required for shearing. If necessary, repeat extraction to achieve required mass for shearing.
Part 2: Shearing with Diagenode Megaruptor 3 Shearing Kit
Part 2: Shearing with Diagenode Megaruptor 3 Shearing Kit
Prepare Amount6-10.5 µg  DNA in Amount150 µL  Buffer EB, at a concentration of 40-70 ng/µL in a Megaruptor 3 shearing tube.

Note: Shearing conditions are optimized for sequencing N50s ~35-45 kb. Shearing with Megaruptor 3 is concentration- and volume-dependent. Samples with a concentration < 40 ng/µL in 150 µL may be susceptible to over-shearing. 
Remove the Megaruptor 3 shearing syringe from the package. Tighten the assembly and ensure the plunger is completely compressed. Attach the syringe to the shearing tube and ensure the syringe is snug on the cap of the tube.
Load samples and syringes onto the Megaruptor 3. If running an odd number of samples, samples can be balanced with an empty corresponding tube.
Run samples on the Megaruptor 3 at speed 20. Enter the appropriate sample volume (150 µL) and sample concentration.
Repeat the previous step for a total of 2 runs on the Megaruptor 3 at speed 20.
Remove the samples from the Megaruptor 3. Remove the syringe from the tube. Make sure the plunger is fully depressed to avoid losing sample volume. Disassemble the needle and remove residual sample from the needle and syringe to avoid losing sample volume.
Transfer entire sample volume to a new 1.5 mL Eppendorf DNA LoBind tube.
Optional: Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA BR Assay. Use Amount1 µL  of DNA per measurement.
Optional: Size on the Agilent Femto Pulse System with the Genomic DNA 165 kb kit. The expected size range for samples post-shearing is 40-100 kb.
Store at Temperature4 °C until ready for size selection. For long-term storage, store at Temperature-80 °C .
Part 3: Removing short DNA fragments with the Short Read Eliminator kit
Part 3: Removing short DNA fragments with the Short Read Eliminator kit
Measure sample volume post-shearing. Use this volume to add Buffer SRE in the next step.

Note: Recommended sample input is 0.6 µg - 9 µg, at 10 ng/µL - 150 ng/µL in 60 µL sample. If necessary, dilute samples with Buffer EB to achieve desired concentration.
Add an equal volume Buffer SRE to each sample. Vortex for Duration00:00:10  and spin down. 
Incubate for Duration01:00:00 at Temperature50 °C in Thermomixer.
Start a centrifuge at 23,100 x g for Duration00:10:00  prior to the end of the previous step.
Load tube into the centrifuge with hinge facing toward the outside of the rotor.

Note: Inserting the tube with the hinge out is crucial to avoid aspirating the pellet in later steps.
 
Figure 3. A. The DNA pellet will form on the side of the tube facing outwards of the rotor, at the bottom of the tube. B. Pipette from the thumb lip side of the tube to avoid disturbing the pellet.

Centrifuge at 10,000 x g for Duration00:30:00  at room temperature.

Note: If using a centrifuge with temperature control, turn this function off by setting the temperature higher than room temperature (29 °C or 30 °C).
Remove tubes from the centrifuge.
Carefully remove supernatant from tube without disturbing the DNA pellet. The DNA pellet will have formed on the bottom of the tube under the hinge region but may not be visible. 
Add Amount55 µL  Buffer LTE.
Note: Buffer volume may be adjusted to achieve desired concentration.
Incubate for Duration00:20:00 at room temperature.

Pipette-mix sample 20x and vortex the tube for Duration00:00:05 . Spin down.

Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA BR Assay. UseAmount2 µL of DNA per measurement.

Note: If necessary, pipette-mix the sample 10x to homogenize before quantification.
Size on the Agilent Femto Pulse System with the Genomic DNA 165 kb kit. The expected size range for samples post-size selection is 40-100kb, with a DQN > 9.8.
Figure 4. Femto Pulse trace post-size selection

Store at Temperature4 °C  until ready for library preparation. At least Amount2.5 µg  DNA is required for library preparation. If necessary, repeat shearing and/or size selection to achieve required mass for library preparation.
Part 4a: Ligation sequencing DNA V14 (SQK-LSK114) library preparation
Part 4a: Ligation sequencing DNA V14 (SQK-LSK114) library preparation
Note: For automated library preparation, see Part 4b: Ligation sequencing DNA V14 (SQK-LSK114) library preparation on Hamilton NGS STAR.
Thaw the following reagents at room temperature, then store on ice:
  • NEBNext FFPE DNA Repair Buffer
  • Ultra II End-Prep Reaction Buffer
  • Ligation Buffer (LNB)
  • Elution Buffer (EB)

Place the following reagents at room temperature:
  • AMPure XP beads
  • Short Fragment Buffer (SFB)
  • Qubit 1X HS dsDNA working solution and standards NOTE: Qubit 1X HS dsDNA working solution must be stored away from light
Prepare Amount2.5 µg DNA in  Amount48 µL  nuclease-free water in a 0.2 mL thin-walled PCR tube.

Note: If necessary, input volume may be greater than 48 µL to accommodate 2.5 µg DNA input mass. Input volume should not exceed 168 µL.
Prepare the following DNA repair enzyme master mix, adjusting the reagent volumes for the number of samples being processed:
  • Amount3.5 µL  NEBNext FFPE DNA Repair Buffer (vortex and spin down)
  • Amount3.5 µL  Ultra II End-Prep Reaction Buffer (vortex and spin down)
  • Amount2 µL  NEBNext FFPE DNA Repair Mix (do not vortex, spin down)
  • Amount3 µL  Ultra II End-Prep Enzyme Mix (do not vortex, spin down)

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep NEB enzyme mixes in the freezer until use and return promptly.

Note: Prepare the DNA repair enzyme master mix with a 10% overage.
Add Amount12 µL  DNA repair enzyme master mix. Pipette-mix or flick mix 10x, and spin down.
Incubate samples at Temperature20 °C  for Duration00:30:00 , followed byTemperature65 °C  for Duration00:05:00 in a thermocycler.

Note: Start and pause thermocycler to allow lid to come to 85 °C before putting samples in.
Transfer samples to new 1.5mL Eppendorf DNA LoBind tubes.
Resuspend AMPure XP beads by vortexing.
Add Amount60 µL  or equal sample volume of resuspended AMPure XP beads. Flick mix 10x and spin down.

Note: AMPure XP bead volume should be adjusted to match the input sample volume and enzyme mix post-incubation. 
Incubate on platform rocker forDuration00:05:00 at room temperature.
Prepare Amount500 µL  of fresh 80% ethanol per sample in nuclease-free water.
Spin down samples and place on magnetic rack. Wait until supernatant is clear and colorless, about Duration00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, add Amount200 µL  of 80% ethanol. Pipette on the opposite wall to avoid disturbing the pellet. After Duration00:00:05 , remove the ethanol. Do not resuspend the beads in ethanol.

Note: If initial volume of beads was significantly higher than 60 µL, more ethanol may be required to keep the beads fully covered.
Repeat the previous step.
Spin down and place tubes on the magnetic rack. Pipette off any residual ethanol.
Allow to dry for ~Duration00:00:30 , but do not overdry to the point of cracking. 
Remove tubes from the magnetic rack. Add Amount60 µL nuclease-free water. Flick mix 10x and spin down.
Incubate forDuration00:03:00  at Temperature37 °C  and 450 rpm in Thermomixer.
Spin down and place the samples on the magnetic rack until eluate is clear and colorless, about Duration00:02:00 .
Transfer Amount60 µL  of eluate into a new 1.5 mL Eppendorf DNA LoBind tube. 

Note: It is possible to store samples at Temperature4 °C  overnight at this step.
Prepare the following ligation enzyme master mix, adjusting the reagent volumes for the number of samples being processed:
  • Amount25 µL Ligation Buffer (pipette-mix, spin down)
  • Amount10 µL Quick T4 DNA Ligase (do not vortex, spin down)
  • Amount5 µL  Ligation Adapter (do not vortex, spin down)

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep Quick T4 DNA Ligase and Ligation Adapter in the freezer until use and return promptly.

Note: Prepare the ligation enzyme master mix with a 10% overage.
Add Amount40 µL  ligation enzyme master mix. Pipette-mix or flick mix 10x, and spin down.
Incubate the samples for Duration00:30:00  at room temperature.
Resuspend AMPure XP beads by vortexing.
Add Amount45 µL  of resuspended AMPure XP beads. Flick mix 10x and spin down.
Incubate on platform rocker for Duration00:05:00  at room temperature.
Spin down samples and place on magnetic rack. Wait until supernatant is clear and colorless, about Duration00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, add Amount250 µL  of Short Fragment Buffer. Remove samples from the magnetic rack, flick mix until beads are fully resuspended, and spin down. 

Wait until supernatant is clear and colorless, about Duration00:02:00 , then remove the supernatant.
Repeat the previous step.
Spin down and place tubes on the magnetic rack. Pipette off any residual Short Fragment Buffer.
Allow to dry for ~Duration00:00:30 , but do not overdry to the point of cracking.
Remove tubes from the magnetic rack. Add Amount26 µL  Elution Buffer. Flick mix 10x and spin down.
Incubate for Duration00:20:00 at Temperature37 °C and 450 rpm in Thermomixer.
Spin down and place the samples on the magnetic rack until eluate is clear and colorless, about Duration00:02:00 .
Transfer Amount26 µL  of eluate into a new 1.5 mL Eppendorf DNA LoBind tube. 
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1X dsDNA HS Assay. Use Amount1 µL of DNA per measurement.
Store at Temperature4 °C  until ready for loading. At least Amount750 ng  DNA is required for loading for a 72 hour run. If necessary, repeat library preparation with additional sheared and size selected DNA to achieve required mass for loading. For long-term storage, store libraries at Temperature-80 °C  .
Note: Recommended DNA input per load is 20 fmol, calculated to be Amount300 ng of 24kb dsDNA using https://nebiocalculator.neb.com/#!/dsdnaamt. For 3 full loads, Amount900 ng  DNA is required. At a minimum, Amount750 ng is required for 2 full loads of Amount300 ng  each, followed by an incomplete load comprised of the recovered DNA library from the first load and the remaining DNA library under 300 ng.

Note: If greater than Amount900 ng  DNA library is available, PromethION flow cell may be loaded 4 times in a 96 hour run for improved data output.
Part 4b: Ligation sequencing DNA V14 (SQK-LSK114) library preparation on Hamilton NGS STAR
Part 4b: Ligation sequencing DNA V14 (SQK-LSK114) library preparation on Hamilton NGS STAR
Note: For manual library preparation, see Part 4a: Ligation sequencing DNA V14 (SQK-LSK114) library preparation.

Thaw the following reagents at room temperature, then store on ice:
  • NEBNext FFPE DNA Repair Buffer
  • Ultra II End-Prep Reaction Buffer
  • Ligation Buffer (LNB)
  • Elution Buffer (EB)
Place the following reagents at room temperature:
  • AMPure XP beads
  • Short Fragment Buffer (SFB)
  • Qubit 1X HS dsDNA working solution and standards NOTE: Qubit 1X HS dsDNA working solution must be stored away from light
Prepare Amount2.5 µg  DNA in  Amount48 µL nuclease-free water in a BioRad Hard-Shell PCR Plate.

Note: DNA input mass should be Amount2 µg  at a minimum. Input volume may not be greater than Amount48 µL . 

Note: Samples must be processed in multiples of 8. If preparing less samples than a multiple of 8, fill remaining wells with Amount48 µL  nuclease-free water.
Launch the LSK-114 protocol in the Hamilton Method Manager.
Note: Ensure the deck layout of the NGS STAR matches the protocol being run. 

Note: Daily and weekly maintenance must be performed prior to beginning the protocol. Follow manufacturer guidelines for preventive maintenance and cleaning procedures. 
Select "Process01: DNA repair and end-prep" as the start process.
Figure 5. Select start process

Select "Process02: DNA repair and end-prep clean-up" as the stop process.
Figure 6. Select stop process

Select the input file worklist.
Figure 7. Select input file worklist

Note: Samples must be processed in multiples of 8. If preparing less samples than a multiple of 8, fill remaining wells with Amount48 µL  nuclease-free water.
Prepare the DNA repair enzyme mix in 2 mL Sarstedt tubes according to the instructions in the method. Avoid pipetting bubbles.
Figure 8. Prepare DNA repair enzyme mix

Load PCR ComfortLids onto the NGS STAR deck according to the instructions in the method.
Figure 9. Load PCR ComfortLids

Load MIDI plates and HSP plates onto the NGS STAR deck according to the instructions in the method.
Figure 10. Load MIDI and HSP plates

Load 50 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 50 µL tips.
Figure 11. Load 50 µL tips

Figure 12. Select loaded 50 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load 300 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 300 µL tips.
Figure 13. Load 300 µL tips

Figure 14. Select loaded 300 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load 80% ethanol in a 60 mL reagent trough in the reagent trough carrier according to the instructions in the method.
Figure 15. Load 80% ethanol

Load nuclease-free water in a 60 mL reagent trough and AMPure XP beads in a 20 mL reagent trough in the reagent trough carrier according to the instructions in the method.
Figure 16. Load nuclease-free water and AMPure XP beads

Note: AMPure XP beads must be resuspended by vortexing prior to use. 
Load 1000 µL tips and the prepared sample plate with Amount48 µL  DNA per well onto the NGS STAR deck according to the instructions in the method. Use the tip count screen to input loaded 1000 µL tips.
Figure 17. Load 1000 µL tips and DNA sample plate

Figure 18. Select loaded 1000 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.

Note: Ensure the magnetic stand is placed on the carrier in the 5th position.
Load the DNA repair enzyme mix tubes onto the CPAC cooler according to the instructions in the method.
Figure 19. Load DNA repair enzyme mix

Note: Enzyme mixes should be spun down and placed on the NGS STAR deck with the caps removed. Take care to avoid pipetting bubbles in the tubes to avoid inaccurate dispensing.
Close the NGS STAR door and press 'Ok.' The protocol will now begin.
When prompted at the end of the run, remove the sample plate from the NGS STAR. Discard the reagent troughs and enzyme mix tubes.
Figure 20. Run completion dialog

Note: It is possible to store samples at Temperature4 °C  overnight at this step.
Launch the LSK-114 protocol in the Hamilton Method Manager.

Note: Ensure the deck layout of the NGS STAR matches the protocol being run. 

Note: Daily and weekly maintenance must be performed prior to beginning the protocol. Follow manufacturer guidelines for preventive maintenance and cleaning procedures. 
Select "Process03: Adapter ligation" as the start process.
Figure 21. Select start process

Select "Process04: Adapter ligation clean-up" as the stop process.
Figure 22. Select stop process

Select the input file worklist.
Figure 23. Select input file worklist

Note: Samples must be processed in multiples of 8. If preparing less samples than a multiple of 8, fill remaining wells with Amount60 µL  nuclease-free water.
Prepare the ligation enzyme mix in 2 mL Sarstedt tubes according to the instructions in the method. Avoid pipetting bubbles.
Figure 24. Prepare ligation enzyme mix

Load MIDI plates and HSP plates onto the NGS STAR deck according to the instructions in the method.
Figure 25. Load MIDI and HSP plates

Load 50 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 50 µL tips.
Figure 26. Load 50 µL tips

Figure 27. Select loaded 50 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load 300 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 300 µL tips.
Figure 28. Load 300 µL tips

Figure 29. Select loaded 300 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load AMPure XP beads in a 20 mL reagent trough, Short Fragment Buffer in a 60 mL reagent trough, and Elution Buffer in a 60 mL reagent trough in the reagent trough carrier according to the instructions in the method.
Figure 30. Load AMPure XP beads, Short Fragment Buffer, and Elution Buffer

Note: AMPure XP beads must be resuspended by vortexing prior to use. 
Load 1000 µL tips and the prepared sample plate with Amount60 µL  DNA per well onto the NGS STAR deck according to the instructions in the method. Use the tip count screen to input loaded 1000 µL tips. 
Figure 31. Load 1000 µL tips and DNA sample plate

Figure 32. Select loaded 1000 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.

Note: Ensure the magnetic stand is placed on the carrier in the 5th position.
Load the ligation enzyme mix tubes onto the CPAC cooler according to the instructions in the method.
Figure 33. Load ligation enzyme mix

Note: Enzyme mixes should be spun down and placed on the NGS STAR deck with the caps removed. Take care to avoid pipetting bubbles in the tubes to avoid inaccurate dispensing.
Close the NGS STAR door and press 'Ok.' The protocol will now begin.
When prompted at the end of the run, remove the sample plate from the NGS STAR. Discard the reagent troughs and enzyme mix tubes.
Figure 34. Run completion dialog

TransferAmount26 µL  of eluate into a new 1.5 mL Eppendorf DNA LoBind tube. 
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1X dsDNA HS Assay. Use Amount1 µL of DNA per measurement.
Store at Temperature4 °C  until ready for loading. At least Amount750 ng  DNA is required for loading. If necessary, repeat library preparation with additional sheared and size selected DNA to achieve required mass for loading. For long-term storage, store libraries at Temperature-80 °C .

Note: Recommended DNA input per load is 20 fmol, calculated to be Amount300 ng of 24kb dsDNA using https://nebiocalculator.neb.com/#!/dsdnaamt. For 3 full loads,Amount900 ng  DNA is required. At a minimum, Amount750 ng  DNA is required for 2 full loads of Amount300 ng  each, followed by an incomplete load comprised of the recovered DNA library from the first load and the remaining DNA library under 300 ng.

Note: If greater than Amount900 ng DNA library is available, PromethION flow cell may be loaded 4 times in a 96 hour run for improved data output.
Part 5: Loading the PromethION Flow Cell
Part 5: Loading the PromethION Flow Cell
Thaw the following reagents at room temperature: 
  • Sequencing Buffer (SB)
  • Library Solution (LIS)
  • Flow Cell Tether (FCT)
  • Flow Cell Flush (FCF)
  • Elution Buffer (EB)

Mix by vortexing, spin down, and place on ice.
Take flow cells out of the fridge. Allow to sit at room temperature for Duration00:20:00 .

Note: Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
Load flow cells into the PromethION docking ports. Perform flow cell check prior to flow cell priming.

Note: Only flow cells with a starting pore count greater than 6500 pores should be used for sequencing runs. Flow cells with starting pore counts less than 6500 pores can be used to generate additional sequencing data after an initial run.
Prepare the flow cell priming mix by combining Amount1170 µL Flow Cell Flush andAmount30 µL  Flow Cell Tether per sample. Mix by vortexing.
Prepare Amount300 ng  DNA library in Amount32 µL Elution Buffer in a 1.5 mL Eppendorf DNA LoBind tube.

Note: Recommended DNA input is 20 fmol, calculated to be 300 ng of 24kb dsDNA using https://nebiocalculator.neb.com/#!/dsdnaamt.
Add Amount100 µL  Sequencing Buffer (SB).
Add Amount68 µL  Library Solution (LIS).
Slide the flow cell inlet port cover clockwise to open. Draw back a small volume to remove any air bubbles:
  • Set a P1000 pipette tip to 200 µL
  • Insert the tip into the inlet port
  • Turn the wheel until dial shows 220-230 µL, until a small volume of buffer enters the pipette tip
Note: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µL, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
Load Amount500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Wait Duration00:05:00 .
Load Amount500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down 5x just prior to loading.
Load Amount200 µL  of library into the inlet port using a P1000 pipette.
Close the valve to seal the inlet port. Install the light shield. Close the PromethION door.
Wait Duration00:10:00  before initiating sequencing run in MinKNOW:
  • Navigate to the start page and click "Start sequencing"
  • Fill in the experiment name and sample ID, select the flow cell position, and load run configuration preset (if applicable)
  • Select the sequencing kit used in the library preparation (SQK-LSK114-XL) on the Kit page
  • Configure sequencing and output parameters - Fast basecalling model, 72 or 96 hour run time, basecalled output off, raw reads output .POD5, and minimum Q score of 8
  • Click "Start" to initiate the sequencing run
Within the first hour of sequencing, pay attention to pore occupancy and pore scan results. If necessary to resolve sequencing issues related to sample or flow cell quality:
  1. Perform pore scan on flow cell and note if it resolves pore count issues.
  2. Stop the sequencing run, remove the flow cell from the PromethION, and insert it again into a different position. Restart the run and note if it resolves pore count issues.
  3. Stop the sequencing run. Recover DNA library and load onto a new flow cell, following protocol from step 117.
Following 72 hours of sequencing and 3 loads of prepared DNA libraries, the sample should yield a data output ~90-100 Gb with an N50 ~35-40 kb.
Figure 35. Read length distribution for blood sample with a 72 hour sequencing run

Figure 36. PromethION pore scan results for blood sample with a 72 hour sequencing run and 3 loads of DNA library

Part 6: Washing and reloading the PromethION Flow Cell
Part 6: Washing and reloading the PromethION Flow Cell
Thaw the following reagents at room temperature: 
  • Sequencing Buffer (SB)
  • Library Solution (LIS)
  • Flow Cell Tether (FCT)
  • Flow Cell Flush (FCF)
  • Elution Buffer (EB)
  • Wash Diluent (DIL)

Mix by vortexing, spin down, and place on ice.
Prepare the flow cell wash mix by combining Amount398 µL  Wash Diluent and Amount2 µL  Wash Mix per sample. Mix by pipetting. Do not vortex. 

Note: Keep Wash Mix (WMX) in the freezer until use and return promptly. Do not vortex.

Note: Prepare the flow cell wash mix with a 10% overage.
Pause the sequencing run in MinKNOW.
If necessary, recover the already loaded DNA library:
  • Slide the inlet port cover clockwise to open
  • Set a P1000 pipette tip to 200 µL
  • Insert the tip into the inlet port
  • Turn the wheel until dial shows 400 µL

Note: Do not attempt to recover the loaded library after removing waste from the flow cell to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels. 

Note: DNA library recovery is necessary for samples with less than Amount900 ng of available DNA library for loading, and can be used for the third load in place a complete Amount300 ng DNA library load.
Remove waste buffer from flow cell:
  • Close the inlet port
  • Insert a P1000 pipette into waste port 2 or 3 and remove the waste buffer
Note: It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Slide the inlet port cover clockwise to open. Draw back a small volume to remove any air bubbles:
  • Set a P1000 pipette tip to 200 µL
  • Insert the tip into the inlet port
  • Turn the wheel until dial shows 220-230 µL, until a small volume of buffer enters the pipette tip
Note: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µL, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
Load Amount400 µL  of the flow cell wash mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Close the inlet port and ensure the light shield is installed.
Wait Duration01:00:00 .
Prepare the flow cell priming mix by combining Amount1170 µL Flow Cell Flush andAmount30 µL  Flow Cell Tether per sample. Mix by vortexing.
Prepare Amount300 ng  DNA library in Amount32 µL Elution Buffer in a 1.5 mL Eppendorf DNA LoBind tube.

Note: Recommended DNA input is 20 fmol, calculated to be 300 ng of 24kb dsDNA using https://nebiocalculator.neb.com/#!/dsdnaamt.

Note: If available sample mass for the third load is under 300 ng, DNA library should be recovered for the first load and used for the third load. Additional DNA library can be spiked in to the recovered DNA library.
Add Amount100 µL  Sequencing Buffer (SB).
Add Amount68 µL  Library Solution (LIS).
Slide the inlet port cover clockwise to open. Draw back a small volume to remove any air bubbles:
  • Set a P1000 pipette tip to 200 µL
  • Insert the tip into the inlet port
  • Turn the wheel until dial shows 220-230 µL, until a small volume of buffer enters the pipette tip
Note: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µL, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
Load Amount500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Wait Duration00:05:00 .
Load Amount500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down 5x just prior to loading.
Load Amount200 µL  of library into the inlet port using a P1000 pipette.
Close the valve to seal the inlet port. Install the light shield. Close the PromethION door.
Wait Duration00:10:00  before resuming sequencing run in MinKNOW.
Within the first hour of sequencing, pay attention to pore occupancy and pore scan results. If necessary to resolve sequencing issues related to sample or flow cell quality:
  1. Perform pore scan on flow cell and note if it resolves pore count issues.
  2. Stop the sequencing run, remove the flow cell from the PromethION, and insert it again into a different position. Note if it resolves pore count issues.
  3. Stop the sequencing run. Recover DNA library and load onto a new flow cell, following protocol from step 139.
Following 72 hours of sequencing and 3 loads of prepared DNA libraries, the sample should yield a data output ~90-100 Gb with an N50 ~35-40 kb.
Figure 37. Read length distribution for blood sample with a 72 hour sequencing run

Figure 38. PromethION pore scan results for blood sample with a 72 hour sequencing run and 3 loads of DNA library

Protocol references
Extracting HMW DNA using Nanobind HT 1 mL blood kit for mammalian whole blood on KingFisher Apex system (PacBio): https://www.pacb.com/wp-content/uploads/Procedure-checklist-Extracting-HMW-DNA-using-the-Nanobind-HT-1-mL-blood-kit-for-human-whole-blood-on-the-KingFisher-Apex-system.pdf

Megaruptor 3 User Guide (Diagenode): https://www.diagenode.com/files/products/shearing_technology/megaruptor/megaruptor-3-manual.pdf

Removing short DNA fragments with the Short Read Eliminator kit (PacBio): https://www.pacb.com/wp-content/uploads/Procedure-checklist-Removing-short-DNA-fragments-with-the-Short-Read-Eliminator-SRE-kit.pdf

Ligation sequencing DNA V14 (SQK-LSK114) library preparation (Oxford Nanopore): https://nanoporetech.com/document/genomic-dna-by-ligation-sqk-lsk114?device=PromethION

Washing and reloading the PromethION Flow Cell (Oxford Nanopore): https://nanoporetech.com/document/flow-cell-wash-kit-exp-wsh004