For optimal performance, NEB recommend the following:

1. Thaw all reagents on ice.

2. Flick and/or invert the reagent tubes to ensure they are well mixed.
   Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.

3. Always spin down tubes before opening for the first time each day.

4. The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
   Note: It is important the buffers are mixed well by vortexing.

5. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

Users can choose between running:

• Full: the complete NBD protocol
• EndRepair: starting from sample input perform only end-repair, barcoding, pooling and clean-up.
• Ligation: starting from barcoded sample pool(s) perform native adapter ligation and clean-up.

Note: For new users we recommend performing full runs.

Additional details on pooling and barcoding by worklist rules can be found in the overview section of this protocol.

If yes, the PDF instructions will open on the PC display.

PDF instructions can also be downloaded: here

A user message will appear prompting worklist file selection. Click 'Next page' to open the pooling worklist folder:

The dropdown menu of pre-prepared csv files found in the worklist folder will open. Scroll through the list using the TouchTools™ display to select the desired option for your run:

Note: The exact directory used may vary between installations of protocol. Training provided by your Tecan representative during installation will specify the location of NBD pooling or barcoding worklist files.

Perform a final review of the selection and variable for your desired method. Click 'Continue' to proceed:

• The MinION option is valid for both the MinION and GridION device.
• We recommend a manual QC step following the native barcoding, pooling and clean-up steps. Please note, this will require the user interaction ~2 hours into the run.
• Barcode allocation can be controlled via a worklist. If required, additional details on pooling and barcoding by worklist rules can be found in the overview section of this protocol.

If 'Barcode addition by worklist: no'

• Starting barcode index defined as alphanumeric coordinate.
• The defined coordinate will be delivered to the sample found in position A01.
• All barcodes after the index given will be delivered to samples sequentially.

For example: If starting barcode set as B03:

• Sample in A01 will recieve barcode B03 (NB11)
• Sample in A02 will recieve barcode B04 (NB12)

Note: If there are too few barcodes provided for the number of samples, this will result in an error and the user will have to re-enter the variables. If running 96 samples, the starting barcode index must be A01.

If 'Barcode addition by worklist: yes'

• Instructions for 'Barcoding worklist input file' will appear on-screen. Click 'Continue' to proceed.

• The dropdown menu of pre-prepared csv files found in the barcode worklist 'file selection' folder will open. Scroll through the list using the TouchTools™ display to select the desired option for your run. Click 'Continue' to proceed.

Note: The exact directory used may vary between installations of protocol. Training provided by your Tecan representative during installation will specify the location of NBD pooling or barcoding worklist files.

Confirmation of final parameter choices:

Option to change variables choices:

Note: If 'Change' is selected this will return user to beginning of script set-up.

If the starting index for the barcoding plate is incompatible with the number of samples, users will be prompted to re-check their set up as follows:

• Keep 'no' selected to keep the default setting for the run.
• Only select 'yes' if you are an experienced user and have a self-validated method.

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• Ensure the master mix is homogenous by pipette mixing.
• Avoid the introduction of air bubbles while preparing the master mix.
• Avoid leaving droplets on the tube wall.
• Briefly spin down the End-prep (EP) master mix to ensure all the liquid is at the bottom of the Sarstedt tube.

Reagent volumes for all sample numbers:

Reagent	Volume X6 samples	Volume X24 samples	Volume X48 samples	Volume X96 samples
NEBNext FFPE DNA Repair Buffer	6.825 µl	27.3 µl	52.5 µl	100.8 µl
Ultra II End-prep reaction buffer	6.825 µl	27.3 µl	52.5 µl	100.8 µl
Ultra II End-prep enzyme mix	5.85 µl	23.4 µl	45 µl	86.4 µl
NEBNext FFPE DNA Repair Mix	3.9 µl	15.6 µl	30 µl	57.6 µl
Total	23.4 µl	93.6 µl	180 µl	345.6 µl

Note: Reagent volumes will vary in accordance with the number of samples selected for processing (6-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• Ensure the master mix is homogenous by pipette mixing.
• Avoid the introduction of air bubbles while preparing the master mix.
• Avoid leaving droplets on the tube wall.
• Briefly spin down the Blunt/TA Ligase master mix to ensure all the liquid is at the bottom of the Sarstedt tube.

Reagent volumes for all sample numbers:

Reagent	Volume X6 samples	Volume X24 samples	Volume X48 samples	Volume X96 samples
NEB Blunt/TA Ligase Master Mix	336 µl	114 µl	264 µl	528 µl

Note: Reagent volumes will vary in accordance with the number of samples selected for processing (6-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• Ensure the master mix is homogenous by pipette mixing.
• Avoid the introduction of air bubbles while preparing the master mix.
• Avoid leaving droplets on the tube wall.
• Briefly spin down the EDTA to ensure all the liquid is at the bottom of the Sarstedt tube.

Reagent volumes for all sample numbers:

Reagent	Volume X6 samples	Volume X24 samples	Volume X48 samples	Volume X96 samples
EDTA	35.6 µl	82.4 µl	135.2 µl	231.2 µl

Note: Reagent volumes will vary in accordance with the number of samples selected for processing (6-96). If processing a different sample input numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

We recommend master mixes are prepared fresh following the instructions below and loaded onto the workspace as soon as possible for optimal results.

• It is essential that no air bubbles are introduced while preparing the Adapter-ligation (AL) master mix.
• Additionally, it is essential that no droplets are left on the tube wall.
• Ensure the master mix is homogenous by carefully pipette mixing. Use the pipette to ensure the all the contents is at the bottom of the tube.

Note: Avoid spinning down the Adapter-ligation (AL) master mix in a centrifuge. This can lead to the reagents in the mix separating, resulting in sub-optimal results.

Reagent volumes for different sample pool numbers:

Reagent	Volume X1 sample pool	Volume X4 sample pool	Volume X8 sample pool	Volume X12 sample pool	Volume X16 sample pool
NEBNext Quick Ligation Reaction Buffer (5X)	18 µl	52 µl	109 µl	149 µl	181 µl
NEBNext Quick T4 DNA Ligase	9 µl	26 µl	54.5 µl	74.5 µl	90.5 µl
Native Adapter (NA)	9 µl	26 µl	54.5 µl	74.5 µl	90.5 µl
Total volume	36 µl	104 µl	218 µl	298 µl	362 µl

Note: Reagent volumes will vary in accordance with the number of sample pools selected for processing (1-16). If processing a different sample pooling numbers, follow the instructions provided by the on-screen display for the correct reagent volumes. Volumes indicated in the table and the on-screen display will include the necessary dead volume excess.

• Ensure the master mixes are thoroughly mixed before loading.
• You will need to select each loading position using the Tecan's TouchTools touchscreen display.
• Follow the instructions on the display for each reagent, ensuring the fill volume for each tube is correct.
• Ensure the reagents have been added to all of the required positions before proceeding.
• Click 'Confirm' to proceed.

On-screen abbreviation glossary:

• End-prep master mix – EP
• Blunt/TA Ligase master mix – B/TA
• EDTA – EDTA
• Adapter ligation master mix – AL

• The required loading position for each box will flash to indicate where to place the labware.
• Remove the tip box lid and load the tip box in the required position.
• After loading all of the required labware, close the front safety shield and click 'Approve' to proceed.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.

• The required loading position for each box will flash to indicate where to place the labware.
• Partially full boxes are supported for the Flexible Channel Arm filtered tips. However, please ensure the tip box contains the minimum required number of tips as described in the equipment and consumables section of this protocol.
• Click 'Approve' after each addition of labware to proceed to the next box.
• After loading all of the required labware, click 'Next Page' to proceed.

Incorrect positioning of the reaction plates will result in incorrect sample tracking throughout the run. Please ensure the reaction plates are placed on the worktable in the correct orientation:

• For the reaction plate(s) loaded in the 'Hotel': The lettered well markers (A-H) should be positioned towards the back of the worktable and the numbered well markers (1-12) should be positioned facing the right.

• For the reaction plate(s) loaded onto the worktable: Follow standard plate orientation, with the lettered well markers (A-H) positioned to the left and the numbered well markers (1-12) positioned to towards the back of the worktable. Note: We recommend all plates are labelled before placing on the worktable to ensure correct plate tracking.

• The required loading position for each plate will flash on the on-screen display to indicate where to place the labware.

• Click 'Approve' after each addition of labware to proceed.

• After loading all of the required labware, click 'Approve'.

BioRad plate addition - hotel site:

BioRad plate addition - workbench site:

Abgene 0.8 ml plate addition - workbench site:

• Ensure all the reagents have been thoroughly mixed by vortexing before dispensing into the tubes.
• The required loading position will clearly indicated via the on-screen instructions.
• Click 'Approve' after each addition to proceed.
• After loading all of the tubes, click 'Approve' to proceed.

Note: Follow the on-screen instructions for each specified reagent and corresponding fill volume.

• Ensure all the reagents have been thoroughly mixed by vortexing before dispensing into the troughs.
• The required loading position will flash to indicate where to insert the trough.
• Click 'Approve' after each addition to proceed.
• After loading all of the troughs, click 'Approve' to proceed.

Note: Follow the on-screen instructions for each specified reagent and corresponding fill volume.

• The required loading position will flash to indicate where to place the labware.
• Click 'Approve' after the addition of the metal lid for ODTC to proceed.

Loading the metal lid for ODTC:

Note: Take care to position the metal lid centrally in the recess. Incorrect positioning of the metal lid can lead to run error.

• The required loading position will flash to indicate where to place the labware.
• After loading the waste plate, click 'Approve'.

Loading the waste plate:

• Aliquot 400 ng DNA per sample into each well.
• Make up each sample to 12 µl using nuclease-free water.
• Mix gently by pipetting and spin down.

• The required loading position will flash to indicate where to place the labware.
• After loading the sample plate, click 'Next Page'.

Loading the sample plate:

• Mix the content of the barcoding plate by placing on a plate shaker for 2 minutes.
• Briefly spin down the plate.
• Remove the foil cover entirely by peeling off.

Note: Failure to remove the foil prior to loading on the workbench can result in run error.

• The required loading position will flash to indicate where to place the labware.
• After loading the native barcode plate, click 'Next Page'.

Loading the native barcode plate:

Once the run starts the screen will display the progress bar.

Fragment library length	Flow cell loading amount
Very short (<1 kb)	100 fmol
Short (1-10 kb)	35–50 fmol
Long (>10 kb)	300 ng

Note: If the library yields are below the input recommendations, load the entire library.

If required, we recommend using a mass to mol calculator such as the NEB calculator.

1. Set a P1000 pipette to 200 µl
2. Insert the tip into the priming port
3. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
   

Note: Visually check that there is continuous buffer from the priming port across the sensor array.

1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

1. Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.

2. Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.

Dispose of the used tips in an appropriate container.

Note: Take care not to spill any residual liquid waste when removing from the worktable.

• Wipe the lid with 10% bleach
• Thoroughly rinse the bleach off the lid using ≥80% ethanol or double distilled water (ddH2O) and lint-free wipes
• Allow the lid to air dry

• Remove all Flexible Channel Arm (FCA) hanging tip trays from the FCA standard tip carriers, and discard accordingly.
• Remove all empty MultiChannel Arm (MCA) tip boxes from the worktable and discard accordingly.
• For partially used tip boxes, consider restacking the box with the appropriate tips for the next run.

Note: The MultiChannel Arm (MCA) tip boxes must be fully stacked. Failure to fully stack the MCA tip boxes can result in run error.

The Flow Cell Wash Kit protocol is available on the Nanopore Community.

Instructions for returning flow cells can be found here.