TITLE: Native Barcoding 
AUTHORS: Carlos Goller, Carly Sjogren

[DESCRIPTION]
This protocol describes how to carry out native barcoding of genomic DNA using the Native Barcoding Kit 24 (SQK-NBD112.24). There are 24 unique barcodes available, allowing the user to pool up to 24 different samples in one sequencing experiment. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

[BEFORE_START]
Read about the kit, the system requirements and other general guidelines regarding samples and reagents from this document in detail. Please go through this thoroughly before beginning. 

Native barcoding kit Information and guidelines

[GUIDELINES]
This protocol describes how to carry out native barcoding of genomic DNA using the Native Barcoding Kit 24 (SQK-NBD112.24). There are 24 unique barcodes available, allowing the user to pool up to 24 different samples in one sequencing experiment. It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

Native Barcoding Kit 24 features
This kit is recommended for users who:
wish to multiplex up to 24 samples to reduce price per sample
need a PCR-free method of multiplexing to preserve additional information such as base modifications
want to optimise their sequencing experiment for throughput
require control over read length
would like to utilise upstream processes such as size selection or whole genome amplification

The Native Barcoding Expansion kit (EXP-NBD112) is available to provide enough reagents for 12 reactions depending on how barcodes are used.

For common issues and concerns regarding the protocol and suggestions to resolve the same, go through this following document, which is also available at the end of the protocol.
Issues while conducting the protocol

[STEPS]
SECTION: Preparation Before Starting
1. Prepare for your experiment
SECTION: Preparation Before Starting
1.1. Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
SECTION: Preparation Before Starting
1.2. Ensure you have your sequencing kit, the correct equipment and third-party reagents
SECTION: Preparation Before Starting
1.3. Download the software for acquiring and analysing your data
SECTION: Preparation Before Starting
1.4. Check your flow cell to ensure it has enough pores for a good sequencing run
SECTION: Preparation Before Starting
2. Prepare your library
  

SECTION: Preparation Before Starting
2.1. Repair the DNA, and prepare the DNA ends for adapter attachment
SECTION: Preparation Before Starting
2.2. Ligate Native barcodes supplied in the kit to the DNA ends
SECTION: Preparation Before Starting
2.3. Ligate sequencing adapters supplied in the kit to the DNA ends
SECTION: Preparation Before Starting
2.4. Prime the flow cell, and load your DNA library into the flow cell
SECTION: Preparation Before Starting
3. Sequencing
SECTION: Preparation Before Starting
3.1. Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
SECTION: Preparation Before Starting
3.2. Demultiplex barcoded reads in MinKNOW or the Guppy basecalling, choosing the SQK-NBD112.24 kit option
SECTION: Preparation Before Starting
3.3. Start the EPI2ME software and select a workflow for further analysis (this step is optional)

 

 

SECTION: DNA repair and end-prep
5. Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.

 

 

 

SECTION: DNA repair and end-prep
6. Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing.
SECTION: DNA repair and end-prep
7. In clean 0.2 ml thin-walled PCR tubes, prepare your DNA sample:
For >4 barcodes, aliquot 400 ng  per sample
For ≤4 barcodes, aliquot 1000 ng  per sample

SECTION: DNA repair and end-prep
8. Make up each sample to 12 µL  using nuclease-free water. Mix gently by pipetting and spin down.

SECTION: DNA repair and end-prep
9. Combine the following components per sample:
Between each addition, pipette mix 10 - 20 times.
SECTION: DNA repair and end-prep
9.1. 
 

 

SECTION: DNA repair and end-prep
10. Mix well by pipetting and spin down in a centrifuge.
SECTION: DNA repair and end-prep
11. Using a thermal cycler, incubate at 20 °C  for 5 min and 65 °C  for 5 min .

SECTION: DNA repair and end-prep
12. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: DNA repair and end-prep
13. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: DNA repair and end-prep
14. Add 15 µL  of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.

SECTION: DNA repair and end-prep
15. Incubate on a Hula mixer (rotator mixer) for 5 min at Room temperature .

SECTION: DNA repair and end-prep
16. Prepare 500 µL of fresh 70% ethanol in nuclease-free water.

SECTION: DNA repair and end-prep
17. Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
SECTION: DNA repair and end-prep
18. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Wait for the beads to migrate towards the magnet and form a pellet. Remove the ethanol using a pipette and discard.

SECTION: DNA repair and end-prep
19. Repeat the previous step.
SECTION: DNA repair and end-prep
20. Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 s .

 


SECTION: DNA repair and end-prep
21. Remove the tube from the magnetic rack and resuspend the pellet in 10 µL  nuclease-free water. Spin down and incubate for 2 min at Room temperature .

SECTION: DNA repair and end-prep
22. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: DNA repair and end-prep
23. Remove and retain 10 µL  of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

 

SECTION: DNA repair and end-prep
24. Take forward equimolar mass of samples to be barcoded and pooled forward into the native barcode step. However, at this point it is also possible to store the sample at 4 °C   overnight.

SECTION: Native Barcode Ligation
26. Prepare third party reagents in accordance with manufacturer's instructions, and place on ice.
SECTION: Native Barcode Ligation
27. Thaw the native barcodes at Room temperature . Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.

SECTION: Native Barcode Ligation
28. Select a unique barcode for every sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment.
SECTION: Native Barcode Ligation
29. In clean 0.2 ml thin-walled PCR tubes, add the reagents in the following order per sample: Between each addition, pipette mix 10 - 20 times.

 

SECTION: Native Barcode Ligation
30. Ensure the components are thoroughly mixed by pipetting and spin down briefly.
SECTION: Native Barcode Ligation
31. Incubate for 20 min at Room temperature .

SECTION: Native Barcode Ligation
32. Add 2 µL of EDTA to each tube and mix thoroughly by pipetting and spin down briefly.

 


SECTION: Native Barcode Ligation
33. Pool the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.
 


 



SECTION: Native Barcode Ligation
34. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Native Barcode Ligation
35. Add AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.4X clean.

 

SECTION: Native Barcode Ligation
36. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature .

SECTION: Native Barcode Ligation
37. Prepare 2 mL of fresh 70% ethanol in nuclease-free water.

SECTION: Native Barcode Ligation
38. Spin down the sample and pellet on a magnet for 5 min  . Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.

SECTION: Native Barcode Ligation
39. Keep the tube on the magnetic rack and wash the beads with 700 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Native Barcode Ligation
40. Repeat the previous step.
SECTION: Native Barcode Ligation
41. Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow to dry for ~30 s .
 


SECTION: Native Barcode Ligation
42. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL nuclease-free water.

SECTION: Native Barcode Ligation
43. Incubate for 10 min at 37 °C . Periodically agitate the sample by gently flicking to encourage DNA elution.

SECTION: Native Barcode Ligation
44. Pellet the beads on a magnetic rack until the eluate is clear and colourless.
SECTION: Native Barcode Ligation
45. Remove and retain 35 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Adapter ligation and clean-up
48. Thaw the Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at Room temperature , mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.

SECTION: Adapter ligation and clean-up
49. Spin down the Quick T4 Ligase and the Adapter Mix II H (AMII H), and place on ice.
SECTION: Adapter ligation and clean-up
50. To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at Room temperature , mix by vortexing, spin down and place on ice.

SECTION: Adapter ligation and clean-up
51. To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at Room temperature , mix by vortexing, spin down and place on ice.

SECTION: Adapter ligation and clean-up
52. In a 1.5 ml Eppendorf LoBind tube, mix in the following order:

 

 

SECTION: Adapter ligation and clean-up
53. Ensure the components are thoroughly mixed by pipetting and spin down briefly.
SECTION: Adapter ligation and clean-up
54. Incubate the reaction for 20 min at Room temperature .
 


SECTION: Adapter ligation and clean-up
55. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Adapter ligation and clean-up
56. Add 20 µL of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.

SECTION: Adapter ligation and clean-up
57. Incubate on a Hula mixer (rotator mixer) for 10 min  at Room temperature 

SECTION: Adapter ligation and clean-up
58. Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
SECTION: Adapter ligation and clean-up
59. Wash the beads by adding either 125 µL Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard

SECTION: Adapter ligation and clean-up
60. Repeat the previous step.
SECTION: Adapter ligation and clean-up
61. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 s 
 


SECTION: Adapter ligation and clean-up
62. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL of Elution Buffer (EB).

SECTION: Adapter ligation and clean-up
63. Spin down and incubate for 10 min at 37 °C  . Periodically agitate the sample by gently flicking to encourage DNA elution

SECTION: Adapter ligation and clean-up
64. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min 

SECTION: Adapter ligation and clean-up
65. Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

 

 


SECTION: Priming and loading the SpotON flow cell
67. We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water
SECTION: Priming and loading the SpotON flow cell
68. Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and one tube of Flush Buffer (FB) at Room temperature before mixing the reagents by vortexing and spin down at Room temperature 

SECTION: Priming and loading the SpotON flow cell
69. To prepare the flow cell priming mix, add 30 µL  of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at Room temperature .

SECTION: Priming and loading the SpotON flow cell
70. Open the MinION lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact
  

SECTION: Priming and loading the SpotON flow cell
72. Slide the priming port cover clockwise to open the priming port.
 

SECTION: Priming and loading the SpotON flow cell
73. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few µl):

  

SECTION: Priming and loading the SpotON flow cell
73.1. Set a P1000 pipette to 200 µl
SECTION: Priming and loading the SpotON flow cell
73.2. Insert the tip into the priming port
SECTION: Priming and loading the SpotON flow cell
73.3. Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering the pipette tip
 

SECTION: Priming and loading the SpotON flow cell
74. Load 800 µL  of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min . During this time, prepare the library for loading by following the steps below.
  

SECTION: Priming and loading the SpotON flow cell
75. Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.

 

SECTION: Priming and loading the SpotON flow cell
76. In a new tube, prepare the library for loading as follows:

 

 

SECTION: Priming and loading the SpotON flow cell
77. Complete the flow cell priming:

SECTION: Priming and loading the SpotON flow cell
77.1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
SECTION: Priming and loading the SpotON flow cell
77.2.  Load 200 µL  of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles
  
 



SECTION: Priming and loading the SpotON flow cell
78. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Priming and loading the SpotON flow cell
79. Add 75 µL  of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
  

SECTION: Priming and loading the SpotON flow cell
80. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION lid. 
  

SECTION: Ending the Experiment
82. 
 

SECTION: Ending the Experiment
83. Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.
Instructions for returning flow cells can be foundhere.

 

SECTION: DNA repair and end-prep
4. Materials 
400 ng gDNA per sample for >4 barcodes 1000 ng gDNA per sample for ≤4 barcodes 
AMPure XP Beads (AXP) 

Consumables 
NEBNext FFPE DNA Repair Mix (M6630) 
NEBNext Ultra II End repair / dA-tailing Module (E7546) 
Nuclease-free water (e.g. ThermoFisher, cat # AM9937) 
Freshly prepared 70% ethanol in nuclease-free water
0.2 ml thin-walled PCR tubes 
1.5 ml Eppendorf DNA LoBind tubes 
Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
P1000 pipette and tips 
P100 pipette and tips
P10 pipette and tips 
Thermal cycler at 20°C and 65°C 
Ice bucket with ice 
Microfuge Hula mixer (gentle rotator mixer) 
Magnetic rack
SECTION: Native Barcode Ligation
25. Materials 
AMPure XP Beads (AXP) 
Native Barcodes (NB01-24) 
EDTA (EDTA)

Consumables 
Nuclease-free water (e.g. ThermoFisher, cat # AM9937) 
Freshly prepared 70% ethanol in nuclease-free water 
NEB Blunt/TA Ligase Master Mix (M0367) 
1.5 ml Eppendorf DNA LoBind tubes 
2 ml Eppendorf DNA LoBind tubes 
0.2 ml thin-walled PCR tubes Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
Magnetic rack 
Vortex mixer 
Hula mixer (gentle rotator mixer) 
Microfuge 
Thermal cycler 
Ice bucket with ice 
P1000 pipette and tips 
P100 pipette and tips 
P10 pipette and tips

Optional Equipment 
Qubit fluorometer (or equivalent for QC check)
SECTION: Native Barcode Ligation
46. Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, at this point it is also possible to store the sample at 4 °C   .

SECTION: Adapter ligation and clean-up
47. Materials 
Long Fragment Buffer (LFB) 
Short Fragment Buffer (SFB) Elution Buffer from the Oxford Nanopore kit (EB) 
Adapter Mix II H (AMII H) 
AMPure XP Beads (AXP)

Consumables 
Quick T4 DNA Ligase in NEBNext® Quick Ligation Module (NEB E6056) 
NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
1.5 ml Eppendorf DNA LoBind tubes 
Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
Microfuge 
Magnetic rack
Vortex mixer 
Hula mixer (gentle rotator mixer) 
Thermal cycler 
P1000 pipette and tips 
P200 pipette and tips 
P100 pipette and tips
P20 pipette and tips 
P10 pipette and tips 
Ice bucket with ice
SECTION: Priming and loading the SpotON flow cell
66. Materials 
Flush Buffer (FB)
Flush Tether (FLT)
Loading Beads II (LBII)
Sequencing Buffer II (SBII)
Loading Solution (LS)

Consumables
1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, cat # AM9937)

Equipment
MinION
SpotON Flow Cell
P1000 pipette and tips
P100 pipette and tips
P20 pipette and tips
P10 pipette and tips
SECTION: Priming and loading the SpotON flow cell
71. Complete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
SECTION: Data Acquisition and Basecalling
81. For information on Data Acquisition and Basecalling, go through the following document in detail
Data Acquisition and Basecalling
SECTION: Issues in the protocol
84. For common issues and concerns regarding the protocol and suggestions to resolve the same, go through this following document, which is also available in guidelines
Issues while conducting the protocol