TITLE: A streamlined protocol for high-throughput amplification-based analysis of DNA samples via nanopore sequencing (based on the 96-well PCR barcoding kit)
AUTHORS: Jordan P Cuff, Annalise S Hooper, Shrinivas  Nivrutti Dighe, Angela Marchbank, Peter  Kille

[DESCRIPTION]
Nanopore sequencing facilitates the rapid and cost-effective sequencing of long fragment DNA for a massive range of applications. When looking to holistically analyse low-yield DNA samples using nanopore sequencing, the optimal method is likely to involve the PCR Barcoding Kit. This effectively involves blunt end ligation of priming sites onto all extant DNA for holistic amplification to achieve yields suitable for nanopore sequencing. The currently available kits from nanopore facilitate the multiplexing of 96 samples in one sequencing run using this method, but the reagent costs are inherently multiplicative. This protocol is designed to streamline (in terms of cost, reagents and time) the process of sequencing up to 96 samples of genomic DNA through nanopore sequencing. 

This protocol is best applied to large numbers of samples (up to 96). For smaller numbers of samples, consider the smaller "PCR Barcoding" kits provided by nanopore which similarly achieve holistic DNA amplification and sequencing, but without the need for additional adapter ligation. The protocol is best suited to samples with low DNA yields (100 ng input is recommended). If you can input 1000 ng of DNA from each of your samples, consider using the 96-well Ligation Kit from Oxford Nanopore which can similarly be streamlined in terms of cost and time, but avoids the amplification step.

[BEFORE_START]
This protocol is best applied to large numbers of samples (up to 96). For smaller numbers of samples, consider the smaller "PCR barcoding" kits provided by nanopore which similarly achieve holistic DNA amplification and sequencing, but without the need for adapter ligation at the end. The protocol is best suited to samples with low DNA yields (100 ng input is recommended). If you can input 1000 ng of DNA from each of your samples, consider using the 96-well ligation kit from Oxford Nanopore which can similarly be streamlined in terms of cost and time, but avoids the amplification step.

[GUIDELINES]
This protocol is designed specifically around the Oxford Nanopore Technologies PCR Barcoding Expansion 1-96 (EXP-PBC096) kit. The kit protocol deviates from the standard PCR barcoding kit protocol, existing as a hybrid between that and the ligation kit (SQK-LSK109). As such, both EXP-PBC096 and SQK-LSK109 will be required (alternative protocols are possible using SQK-LSK110). 

This protocol is designed to streamline (in terms of both cost/reagents and time) the process of sequencing many (up to 96) samples of genomic DNA using nanopore sequencing. The process involves ligating priming sites onto the genomic DNA, priming and amplifying the full DNA strands and then ligating adapters onto the amplified products.

[STEPS]
SECTION: Prepare DNA
1. Extract and purify your DNA in a manner appropriate for the sample type, the hypothesis being tested and this protocol. Consider the length of DNA that you are ideally interested in and how the tissue/sample type might best be lysed/extracted from. This protocol will be most successful when using samples with low concentrations of contaminants/inhibitors.
SECTION: Ligation of barcode adapters
2. Before beginning the ligation process, it would be ideal to quantify the concentration of your samples using Qubit high-sensitivity/broad-range (depending on application) dsDNA assays or similar, but this can be omitted if necessary. 
SECTION: Amplification of libraries
8. LongAmp Hot Start Taq 2X Master Mix, Barcode Primer 1 (BP 01). 

Set up the following reaction:

  VolumeReagent10 µlAdapter-ligated DNA14 µlNuclease-free water1 µlBarcode Primer (BC01-96)25 µl2X LongAmp Taq Master MixTotal: 50 µl 

SECTION: Ligation of adapters
13. Get your adapter ligation reaction ready in the order given:

DNA47 µLDNA CS1 µLNEBNext FFPE DNA Repair Buffer3.5 µLNEBNext FFPE DNA Repair Mix2 µLUltra II End-prep reaction buffer3.5Ultra II End-prep enzyme mix3Total60 µL

SECTION: Loading of library (flongle)
19. If looking to check the quality of the DNA for sequencing, or only looking to generate relatively small amounts of sequencing data, consider sequencing on a flongle.

Allow the flongle to reach room temperature for 20-30 mins ahead of loading of the library. Insert the flongle into the MinION sequencer and check the number of active pores available. Visually check the flongle for air bubbles.

Thaw the sequencing buffer (SQB), loading beads (LB), flush tether (FLT) and flush buffer (FB) at room temperature  and vortex all but the FLT briefly and spin them down.

Put together the following flush solution: 
VolumeReagent3 µlFlush Tether (FLT)117 µlFlush Buffer (FB)Total: 120 µl 


Of this, add 100 μL to the flongle via the priming port, with caution to avoid the introduction of air bubbles.

Ensure the beads of the LB are suspended by vortexing again immediately before pipetting, then assemble the following solution:

  VolumeReagent10 µlLoading Beads (LB)15 µlSequencing Buffer (SQB)5 µlDNA libraryTotal: 30 µl 

Gently mix the library by gentle flicking and inversion immediately before loading 30 μL via the priming port. Begin sequencing using the default settings for 24 hours.
SECTION: Ligation of barcode adapters
3. In a new 0.2 ml tube (or 96-well plate), set up the following reaction for each sample:
 
  ComponentVolumeDNA in nuclease-free water12.5 µLUltra II End Prep Reaction Buffer1.75 µLUltra II End Prep Enzyme Mix0.75 µLTotal15 µL

SECTION: Ligation of barcode adapters
5. Get your barcode adapter ligation reaction ready in the order given:
 
 

SECTION: Ligation of barcode adapters
7. Clean the adapter-ligated DNA with 0.4X AMPure beads (13.2 μL), washing twice with 200 μL 70% ethanol. Resuspend in 26 μL nuclease-free water, leaving 1 μL behind to prevent bead uptake.
SECTION: Amplification of libraries
9. Mix the reaction by gentle flicking and inversion, and incubate as follows:
 
   TemperatureTimeCycles95 °C3 mins195 °C0:15 mins1862 °C0:15 mins65 °C16:40 mins65 °C6 mins14 °C--

 The extension length (V) depends on the expected DNA length. Approximately 50 seconds per kbp is recommended (e.g., 16:40 for expected fragment sizes of ~20 kbp). Sizes can be pre-determined using gel electrophoresis or digital analysis tools such as TapeStation or Qiaxcel.
 
SECTION: Amplification of libraries
10. Measure the concentration of your samples using Qubit high-sensitivity/broad-range (depending on expected concentration) dsDNA assays (or similar).
SECTION: Amplification of libraries
11. Pool your barcoded samples together based on concentration to achieve approximate equimolarity.
SECTION: Amplification of libraries
12. Clean the adapter-ligated DNA with 1X AMPure beads, washing twice with 200 μL 70% ethanol. Resuspend in 50 μL nuclease-free water, taking 48 μL out (leaving 2 μL behind to prevent bead uptake), and using 1 μL to measure concentration.
SECTION: Ligation of adapters
15. Clean the sample with 1X AMPure beads, washing twice with 200 μL 70% ethanol. Resuspend in 61 μL nuclease-free water, taking 60 μL out (leaving 1 μL behind to prevent bead uptake).

You can theoretically skip this clean, but if you do, do not incubate for longer than 10 mins after the next step.
SECTION: Ligation of adapters
16. Get your ligation reaction ready in the order given:

DNA60 µLAdapter Mix AMX5 µLLigation Buffer LNB (or T4 buffer)25 µLNEBNext Quick T4 DNA Ligase10 µLTotal100 µL

SECTION: Ligation of adapters
18. Clean the sample with 1X AMPure beads, washing twice with either LFB or SFB (DO NOT CLEAN WITH ETHANOL; SFB for target fragments less than 3 kbp in length, otherwise use LFB). Resuspend in 17 μL elution buffer (EB, or 10mM Tris-HCl pH 8.0 + 50mM NaCl), leaving 1 μL of it behind and measuring the concentration of a further 1 μL using Qubit or similar.

Target input should be:
5-50 fmol for R9.4.1 flow cell
25-75 fmol for R10.3 flow cells
3-20 fmol for flongle flow cells
SECTION: Loading of library (full flow cell)
20. Allow the flow cell to reach room temperature for 20-30 mins ahead of loading of the library. Insert the flow cell into the MinION sequencer and check the number of active pores available. Visually check the flow cell for air bubbles. Removing a small (<20  ul) amount of yellow buffer to purge any air.

Thaw the sequencing buffer (SQB), loading beads (LB), flush tether (FLT) and a full tube of flush buffer (FB) at room temperature, briefly vortex all but the FLT and spin them down. To the full FB tube, add 30 µl FLT, and mix by gentle flicking and inversion. Of this, add 800 µl to the flow cell via the priming port, with  caution to avoid the introduction of air bubbles.

Ensure the beads of the LB are suspended by vortexing again immediately before pipetting, then assemble the following solution: 
  VolumeReagent25.5 µlLoading Beads (LB)34 µlSequencing Buffer (SQB)4.5 µlNuclease-free water11 µlDNA libraryTotal: 75 µl 

This was gently mix the library by gentle flicking and inversion, load 75 µl via the SpotON sample port in a dropwise manner, ensuring each drop flows into the port before adding the next. The flow cell can then begin sequencing using the default settings.
 
 
 
 
SECTION: Prepare DNA
1.1. Consider whether a size selection/clean/concentration would be appropriate for the samples. If targeting long fragments, clean the samples with a 0.4X SPRI Select bead clean, washing twice with 200 μL 70% ethanol and eluting into 12.5 μL nuclease-free water (the step-by-step protocol for bead cleans is given below in Step 7). If the samples are low concentration or contain a high concentration of inhibitors/contaminants, consider carrying out a 1X bead clean to concentrate them and remove inhibitors.

SECTION: Ligation of barcode adapters
6. Incubate the samples as follows:

 

 
SECTION: Prepare DNA
1.2. Consider running the samples in duplicate/triplicate for best results. Ideally, these would use different barcodes downstream for post-sequencing comparison of results between samples to ascertain consistency.
SECTION: Ligation of barcode adapters
4. Incubate the samples as follows:

 

SECTION: Ligation of barcode adapters
7.1. If unfamiliar with bead cleaning, it will be described once here which can be referred to for subsequent cleans. Stay vigilant regarding the volume/ratio of beads to add, the solution used to wash the beads (i.e., ethanol vs. SFB/LFB) and the resuspension volume.

First, resuspend the beads in solution, ensuring they are at room temperature.
SECTION: Amplification of libraries
11.1. Divide each concentration by the largest concentration across all samples to determine the volume of each to add to  achieve approximate equimolarity.
SECTION: Ligation of adapters
14. Incubate the sample as follows:
 
 

SECTION: Ligation of adapters
17. Incubate at room temperature (~20 °C) for 10 mins.
SECTION: Ligation of barcode adapters
3.1. Consider including negative and positive controls alongside your samples which can be sequenced for later quality control.
SECTION: Ligation of barcode adapters
7.2. Add the required volume of beads (e.g., 0.4X clean of 33 μL sample requires an addition of 13.2 μL bead solution) and mix by gentle flicking and inversion (slower) or pipetting (risks fragmentation).
SECTION: Ligation of barcode adapters
7.3. Incubate the solution for 5 mins (unless not physically mixed, in which case incubate for at least 10 mins). The solution can be incubated on a mixer to ensure thorough mixing.
SECTION: Ligation of barcode adapters
7.4. Prepare the cleaning solution (e.g., make up fresh 70 % ethanol or thaw LFB/SFB).
SECTION: Ligation of barcode adapters
7.5. Place the sample on a magnet for 5 mins to separate the beads from the rest of the solution. 
SECTION: Ligation of barcode adapters
7.6. Keeping the sample on the magnet, pipette off the supernatant and wash the beads with the cleaning solution (e.g., 200 μL 70 % ethanol) without disturbing the pelleted beads.
SECTION: Ligation of barcode adapters
7.7. Incubate for 30 seconds and remove the ethanol supernatant by pipetting without disturbing the pelleted beads. Wash the beads with a further round of the cleaning solution (e.g., 200 μL 70 % ethanol) without disturbing the pelleted beads.
SECTION: Ligation of barcode adapters
7.8. Pipette off any residual cleaning solution (briefly spinning down first can help, but ensure the sample is back on the magnet before attempting to remove any solution). Briefly air dry the beads (waiting for the beads to lose their glossy shimmer and become matte - this is usually ~30-120 seconds for SPRI and AMPure depending on the efficiency with which cleaning solution is removed). 
SECTION: Ligation of barcode adapters
7.9. Resuspend the pellet with the required resuspension volume (e.g., 25 μL nuclease-free water) and incubate for 5 mins. 
SECTION: Ligation of barcode adapters
7.10. Pellet the beads on a magnet until the solution is clear and colourless, and the beads are fully separated. 
SECTION: Ligation of barcode adapters
7.11. Remove and retain the required volume of resuspended DNA in a new tube.