Native Barconding Amplicon

Introduction to the protocol
Overview of the protocol
IMPORTANT
This is a Legacy product
This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit
which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product,
please contact Customer Support via email: [email protected].
Native Barcoding Expansion 1-12 and 13-24 features
These kits are recommended for users who:
wish to multiplex samples to reduce price per sample

need a relatively quick method of multiplexing, avoiding the need of further PCR
want to optimise their sequencing experiment for throughput
Introduction to the Native Barcoding protocol
This protocol describes how to carry out native barcoding of amplicons using the Native Barcoding Expansion 1-12 (EXP-NBD104) and
13-24 (EXP-NBD114), in conjunction with the Ligation Sequencing Kit (SQK-LSK109). There are 24 unique barcodes if using both
expansion kits, 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.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:

- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the software for acquiring and analysing your data
- Check your flow cell to ensure it has enough pores for a good sequencing run
Prepare your library
You will need to:

- Prepare the DNA ends for adapter attachment
- Attach Native barcodes supplied in the kit to the DNA ends
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
End-prep
p
A
p
A
Ligation of T
150 min barcodes
Ligation of
sequencing adapters
Loading
Sequencing
You will need to:

- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
- Start the EPI2ME software and select the barcoding workflow
IMPORTANT
We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.
CAUTION
Read concatemerisation
Please note that we observe a small but significant proportion of reads forming concatemers when using ligation chemistry with
amplicons (5–7% for 1 kb amplicons tested). For this reason, we generally recommend using the PCR Barcoding Expansion Kits
(EXP-PBC001 and EXP-PBC096) for barcoding amplicons. If you do wish to use native barcoding with amplicons, please ensure
your bioinformatic analysis workflow is able to identify such concatemers.
IMPORTANT
Compatibility of this protocol
This protocol should only be used in combination with:
Ligation Sequencing Kit (SQK-LSK109)

Native Barcoding Expansions 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114)
FLO-MIN106 (R9.4.1) flow cells
Flow Cell Wash Kit (EXP-WSH004)
Equipment and consumables

Materials 1 µg (or 100–200 fmol) amplicon DNA for every sample to be barcoded
Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more
than 12 samples
Ligation Sequencing Kit (SQK-LSK109)
Flow Cell Priming Kit (EXP-FLP002)
Adapter Mix II Expansion (EXP-AMII001)
Consumables Agencourt AMPure XP beads (Beckman Coulter™, cat # A63881)

NEB Blunt/TA Ligase Master Mix (NEB, M0367)
NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubes
Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
Freshly prepared 70% ethanol in nuclease-free water
Equipment Hula mixer (gentle rotator mixer)

Magnetic separator, suitable for 1.5 ml Eppendorf tubes
Microfuge
Vortex mixer
Thermal cycler
P1000 pipette and tips
P2 pipette and tips
Ice bucket with ice
Timer
Optional Agilent Bioanalyzer (or equivalent)

Equipment Qubit fluorometer (or equivalent for QC check)
Eppendorf 5424 centrifuge (or equivalent)
For this protocol, you will need 1 µg (or 100-200 fmol) amplicon DNA for every sample to be barcoded:
Input DNA
How to QC your input DNA
It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor
quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read theInput DNA/RNA QC protocol.
Chemical contaminants
Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which
can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the
Community.
Ligation Sequencing Kit contents (SQK-LSK109)
Name Acronym Cap colour No. of vials Fill volume per vial (µl)
DNA CS DCS Yellow 1 50
Adapter Mix AMX Green 1 40
Ligation Buffer LNB Clear 1 200
Long Fragment Buffer LFB White cap, orange stripe on 2 1,800

label
Short Fragment Buffer SFB Grey 2 1,800
Sequencing Buffer SQB Red 2 300
Elution Buffer EB Black 1 200
Loading Beads LB Pink 1 360
Sequencing Tether SQT Purple 1 10
Flow Cell Priming Kit contents (EXP-FLP002)

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Flush Buffer FB Blue 6 1,170
Flush Tether FLT Purple 1 200
Adapter Mix II Expansion contents (EXP-AMII001)
AMII AMII
AMII : Adapter Mix II
Name Acronym Cap colour No. of tubes Fill volume per vial (μl)
Adapter Mix AMII Green 2 40

II
Adapter Mix II Expansion use
Protocols that use the Native Barcoding Expansions require 5 μl of AMII per reaction. Native Barcoding Expansions EXP-
NBD104/NBD114 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 reactions when
sequencing on Flongle). This assumes that all barcodes are used in one sequencing run.
The Adapter Mix II expansion provides additional AMII for customers who are running subsets of barcodes, and allows a further 12
reactions (24 on Flongle).
Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) contents
EXP-NBD104 kit contents
NBD NBD NBD NBD NBD NBD NBD

01 02 03 04 05 06 07
NBD NBD NBD NBD NBD

AMII
08 09 10 11 12
NBD01 : Native barcode 1 NBD08: Native barcode 8

NBD06 : Native barcode 6 AMII : Adapter mix II
NBD07 : Native barcode 7
Native Barcode 01-12 NB01-12 White 12 20
Adapter Mix II AMII Green 1 40
EXP-NBD114 kit contents
NBD NBD NBD NBD NBD NBD NBD

13 14 15 16 17 18 19
NBD NBD NBD NBD NBD

AMII
20 21 22 23 24
NBD13 : Native barcode 13 NBD20 : Native barcode 20

NBD18 : Native barcode 18 AMII : Adapter mix II
NBD19 : Native barcode 19
Native Barcode 13-24 NB13-24 White 12 20
Adapter Mix II AMII Green 1 40
Native barcode sequences
The native barcode sequences are the reverse complement of the corresponding barcode sequence in other kits. The first 24 unique
barcodes are available in the Native Barcoding Kit 24 V14 (SQK-NBD114.24) and the Native Barcoding Expansion 1-12 and 13-24 (EXP-
NBD104 and EXP-NBD114). The Native Barcoding Kit 96 V14 (SQK-NBD114.96) and Native Barcoding Expansion 96 (EXP-NBD196)
include the first 24 native barcodes, with an additional 72 unique barcodes.
Native Barcoding Kit 24 V14 (SQK-NBD114.24) and Native Barcoding Expansion 1-12 and 13-24 (EXP-NBD104 and EXP-
NBD114)
Component Forward sequence Reverse sequence
NB01 CACAAAGACACCGACAACTTTCTT AAGAAAGTTGTCGGTGTCTTTGTG
NB02 ACAGACGACTACAAACGGAATCGA TCGATTCCGTTTGTAGTCGTCTGT
NB03 CCTGGTAACTGGGACACAAGACTC GAGTCTTGTGTCCCAGTTACCAGG
NB04 TAGGGAAACACGATAGAATCCGAA TTCGGATTCTATCGTGTTTCCCTA
NB05 AAGGTTACACAAACCCTGGACAAG CTTGTCCAGGGTTTGTGTAACCTT
NB06 GACTACTTTCTGCCTTTGCGAGAA TTCTCGCAAAGGCAGAAAGTAGTC
NB07 AAGGATTCATTCCCACGGTAACAC GTGTTACCGTGGGAATGAATCCTT
NB08 ACGTAACTTGGTTTGTTCCCTGAA TTCAGGGAACAAACCAAGTTACGT

NB09 AACCAAGACTCGCTGTGCCTAGTT AACTAGGCACAGCGAGTCTTGGTT
NB10 GAGAGGACAAAGGTTTCAACGCTT AAGCGTTGAAACCTTTGTCCTCTC
NB11 TCCATTCCCTCCGATAGATGAAAC GTTTCATCTATCGGAGGGAATGGA
NB12 TCCGATTCTGCTTCTTTCTACCTG CAGGTAGAAAGAAGCAGAATCGGA
NB13 AGAACGACTTCCATACTCGTGTGA TCACACGAGTATGGAAGTCGTTCT
NB14 AACGAGTCTCTTGGGACCCATAGA TCTATGGGTCCCAAGAGACTCGTT
NB15 AGGTCTACCTCGCTAACACCACTG CAGTGGTGTTAGCGAGGTAGACCT
NB16 CGTCAACTGACAGTGGTTCGTACT AGTACGAACCACTGTCAGTTGACG
NB17 ACCCTCCAGGAAAGTACCTCTGAT ATCAGAGGTACTTTCCTGGAGGGT
NB18 CCAAACCCAACAACCTAGATAGGC GCCTATCTAGGTTGTTGGGTTTGG
NB19 GTTCCTCGTGCAGTGTCAAGAGAT ATCTCTTGACACTGCACGAGGAAC
NB20 TTGCGTCCTGTTACGAGAACTCAT ATGAGTTCTCGTAACAGGACGCAA
NB21 GAGCCTCTCATTGTCCGTTCTCTA TAGAGAACGGACAATGAGAGGCTC
NB22 ACCACTGCCATGTATCAAAGTACG CGTACTTTGATACATGGCAGTGGT
NB23 CTTACTACCCAGTGAACCTCCTCG CGAGGAGGTTCACTGGGTAGTAAG
NB24 GCATAGTTCTGCATGATGGGTTAG CTAACCCATCATGCAGAACTATGC
Native Barcoding Kit 96 (SQK-NBD112.96) and Native Barcoding Expansion 96 (EXP-NBD196)
NB01 CACAAAGACACCGACAACTTTCTT AAGAAAGTTGTCGGTGTCTTTGTG
NB02 ACAGACGACTACAAACGGAATCGA TCGATTCCGTTTGTAGTCGTCTGT
NB03 CCTGGTAACTGGGACACAAGACTC GAGTCTTGTGTCCCAGTTACCAGG
NB04 TAGGGAAACACGATAGAATCCGAA TTCGGATTCTATCGTGTTTCCCTA
NB05 AAGGTTACACAAACCCTGGACAAG CTTGTCCAGGGTTTGTGTAACCTT
NB06 GACTACTTTCTGCCTTTGCGAGAA TTCTCGCAAAGGCAGAAAGTAGTC
NB07 AAGGATTCATTCCCACGGTAACAC GTGTTACCGTGGGAATGAATCCTT
NB08 ACGTAACTTGGTTTGTTCCCTGAA TTCAGGGAACAAACCAAGTTACGT
NB09 AACCAAGACTCGCTGTGCCTAGTT AACTAGGCACAGCGAGTCTTGGTT
NB10 GAGAGGACAAAGGTTTCAACGCTT AAGCGTTGAAACCTTTGTCCTCTC
NB11 TCCATTCCCTCCGATAGATGAAAC GTTTCATCTATCGGAGGGAATGGA
NB12 TCCGATTCTGCTTCTTTCTACCTG CAGGTAGAAAGAAGCAGAATCGGA
NB13 AGAACGACTTCCATACTCGTGTGA TCACACGAGTATGGAAGTCGTTCT
NB14 AACGAGTCTCTTGGGACCCATAGA TCTATGGGTCCCAAGAGACTCGTT

NB15 AGGTCTACCTCGCTAACACCACTG CAGTGGTGTTAGCGAGGTAGACCT
NB16 CGTCAACTGACAGTGGTTCGTACT AGTACGAACCACTGTCAGTTGACG
NB17 ACCCTCCAGGAAAGTACCTCTGAT ATCAGAGGTACTTTCCTGGAGGGT
NB18 CCAAACCCAACAACCTAGATAGGC GCCTATCTAGGTTGTTGGGTTTGG
NB19 GTTCCTCGTGCAGTGTCAAGAGAT ATCTCTTGACACTGCACGAGGAAC
NB20 TTGCGTCCTGTTACGAGAACTCAT ATGAGTTCTCGTAACAGGACGCAA
NB21 GAGCCTCTCATTGTCCGTTCTCTA TAGAGAACGGACAATGAGAGGCTC
NB22 ACCACTGCCATGTATCAAAGTACG CGTACTTTGATACATGGCAGTGGT
NB23 CTTACTACCCAGTGAACCTCCTCG CGAGGAGGTTCACTGGGTAGTAAG
NB24 GCATAGTTCTGCATGATGGGTTAG CTAACCCATCATGCAGAACTATGC
NB25 GTAAGTTGGGTATGCAACGCAATG CATTGCGTTGCATACCCAACTTAC
NB26 CATACAGCGACTACGCATTCTCAT ATGAGAATGCGTAGTCGCTGTATG
NB27 CGACGGTTAGATTCACCTCTTACA TGTAAGAGGTGAATCTAACCGTCG
NB28 TGAAACCTAAGAAGGCACCGTATC GATACGGTGCCTTCTTAGGTTTCA
NB29 CTAGACACCTTGGGTTGACAGACC GGTCTGTCAACCCAAGGTGTCTAG
NB30 TCAGTGAGGATCTACTTCGACCCA TGGGTCGAAGTAGATCCTCACTGA
NB31 TGCGTACAGCAATCAGTTACATTG CAATGTAACTGATTGCTGTACGCA
NB32 CCAGTAGAAGTCCGACAACGTCAT ATGACGTTGTCGGACTTCTACTGG
NB33 CAGACTTGGTACGGTTGGGTAACT AGTTACCCAACCGTACCAAGTCTG
NB34 GGACGAAGAACTCAAGTCAAAGGC GCCTTTGACTTGAGTTCTTCGTCC
NB35 CTACTTACGAAGCTGAGGGACTGC GCAGTCCCTCAGCTTCGTAAGTAG
NB36 ATGTCCCAGTTAGAGGAGGAAACA TGTTTCCTCCTCTAACTGGGACAT
NB37 GCTTGCGATTGATGCTTAGTATCA TGATACTAAGCATCAATCGCAAGC
NB38 ACCACAGGAGGACGATACAGAGAA TTCTCTGTATCGTCCTCCTGTGGT
NB39 CCACAGTGTCAACTAGAGCCTCTC GAGAGGCTCTAGTTGACACTGTGG
NB40 TAGTTTGGATGACCAAGGATAGCC GGCTATCCTTGGTCATCCAAACTA
NB41 GGAGTTCGTCCAGAGAAGTACACG CGTGTACTTCTCTGGACGAACTCC
NB42 CTACGTGTAAGGCATACCTGCCAG CTGGCAGGTATGCCTTACACGTAG
NB43 CTTTCGTTGTTGACTCGACGGTAG CTACCGTCGAGTCAACAACGAAAG
NB44 AGTAGAAAGGGTTCCTTCCCACTC GAGTGGGAAGGAACCCTTTCTACT
NB45 GATCCAACAGAGATGCCTTCAGTG CACTGAAGGCATCTCTGTTGGATC
NB46 GCTGTGTTCCACTTCATTCTCCTG CAGGAGAATGAAGTGGAACACAGC

NB47 GTGCAACTTTCCCACAGGTAGTTC GAACTACCTGTGGGAAAGTTGCAC
NB48 CATCTGGAACGTGGTACACCTGTA TACAGGTGTACCACGTTCCAGATG
NB49 ACTGGTGCAGCTTTGAACATCTAG CTAGATGTTCAAAGCTGCACCAGT
NB50 ATGGACTTTGGTAACTTCCTGCGT ACGCAGGAAGTTACCAAAGTCCAT
NB51 GTTGAATGAGCCTACTGGGTCCTC GAGGACCCAGTAGGCTCATTCAAC
NB52 TGAGAGACAAGATTGTTCGTGGAC GTCCACGAACAATCTTGTCTCTCA
NB53 AGATTCAGACCGTCTCATGCAAAG CTTTGCATGAGACGGTCTGAATCT
NB54 CAAGAGCTTTGACTAAGGAGCATG CATGCTCCTTAGTCAAAGCTCTTG
NB55 TGGAAGATGAGACCCTGATCTACG CGTAGATCAGGGTCTCATCTTCCA
NB56 TCACTACTCAACAGGTGGCATGAA TTCATGCCACCTGTTGAGTAGTGA
NB57 GCTAGGTCAATCTCCTTCGGAAGT ACTTCCGAAGGAGATTGACCTAGC
NB58 CAGGTTACTCCTCCGTGAGTCTGA TCAGACTCACGGAGGAGTAACCTG
NB59 TCAATCAAGAAGGGAAAGCAAGGT ACCTTGCTTTCCCTTCTTGATTGA
NB60 CATGTTCAACCAAGGCTTCTATGG CCATAGAAGCCTTGGTTGAACATG
NB61 AGAGGGTACTATGTGCCTCAGCAC GTGCTGAGGCACATAGTACCCTCT
NB62 CACCCACACTTACTTCAGGACGTA TACGTCCTGAAGTAAGTGTGGGTG
NB63 TTCTGAAGTTCCTGGGTCTTGAAC GTTCAAGACCCAGGAACTTCAGAA
NB64 GACAGACACCGTTCATCGACTTTC GAAAGTCGATGAACGGTGTCTGTC
NB65 TTCTCAGTCTTCCTCCAGACAAGG CCTTGTCTGGAGGAAGACTGAGAA
NB66 CCGATCCTTGTGGCTTCTAACTTC GAAGTTAGAAGCCACAAGGATCGG
NB67 GTTTGTCATACTCGTGTGCTCACC GGTGAGCACACGAGTATGACAAAC
NB68 GAATCTAAGCAAACACGAAGGTGG CCACCTTCGTGTTTGCTTAGATTC
NB69 TACAGTCCGAGCCTCATGTGATCT AGATCACATGAGGCTCGGACTGTA
NB70 ACCGAGATCCTACGAATGGAGTGT ACACTCCATTCGTAGGATCTCGGT
NB71 CCTGGGAGCATCAGGTAGTAACAG CTGTTACTACCTGATGCTCCCAGG
NB72 TAGCTGACTGTCTTCCATACCGAC GTCGGTATGGAAGACAGTCAGCTA
NB73 AAGAAACAGGATGACAGAACCCTC GAGGGTTCTGTCATCCTGTTTCTT
NB74 TACAAGCATCCCAACACTTCCACT AGTGGAAGTGTTGGGATGCTTGTA
NB75 GACCATTGTGATGAACCCTGTTGT ACAACAGGGTTCATCACAATGGTC
NB76 ATGCTTGTTACATCAACCCTGGAC GTCCAGGGTTGATGTAACAAGCAT
NB77 CGACCTGTTTCTCAGGGATACAAC GTTGTATCCCTGAGAAACAGGTCG
NB78 AACAACCGAACCTTTGAATCAGAA TTCTGATTCAAAGGTTCGGTTGTT

NB79 TCTCGGAGATAGTTCTCACTGCTG CAGCAGTGAGAACTATCTCCGAGA
NB80 CGGATGAACATAGGATAGCGATTC GAATCGCTATCCTATGTTCATCCG
NB81 CCTCATCTTGTGAAGTTGTTTCGG CCGAAACAACTTCACAAGATGAGG
NB82 ACGGTATGTCGAGTTCCAGGACTA TAGTCCTGGAACTCGACATACCGT
NB83 TGGCTTGATCTAGGTAAGGTCGAA TTCGACCTTACCTAGATCAAGCCA
NB84 GTAGTGGACCTAGAACCTGTGCCA TGGCACAGGTTCTAGGTCCACTAC
NB85 AACGGAGGAGTTAGTTGGATGATC GATCATCCAACTAACTCCTCCGTT
NB86 AGGTGATCCCAACAAGCGTAAGTA TACTTACGCTTGTTGGGATCACCT
NB87 TACATGCTCCTGTTGTTAGGGAGG CCTCCCTAACAACAGGAGCATGTA
NB88 TCTTCTACTACCGATCCGAAGCAG CTGCTTCGGATCGGTAGTAGAAGA
NB89 ACAGCATCAATGTTTGGCTAGTTG CAACTAGCCAAACATTGATGCTGT
NB90 GATGTAGAGGGTACGGTTTGAGGC GCCTCAAACCGTACCCTCTACATC
NB91 GGCTCCATAGGAACTCACGCTACT AGTAGCGTGAGTTCCTATGGAGCC
NB92 TTGTGAGTGGAAAGATACAGGACC GGTCCTGTATCTTTCCACTCACAA
NB93 AGTTTCCATCACTTCAGACTTGGG CCCAAGTCTGAAGTGATGGAAACT
NB94 GATTGTCCTCAAACTGCCACCTAC GTAGGTGGCAGTTTGAGGACAATC
NB95 CCTGTCTGGAAGAAGAATGGACTT AAGTCCATTCTTCTTCCAGACAGG
NB96 CTGAACGGTCATAGAGTCCACCAT ATGGTGGACTCTATGACCGTTCAG
Computer requirements and software
MinION Mk1B IT requirements
Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. Read more in the
MinION IT Requirements document.
MinION Mk1C IT requirements
The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse
nanopore data. Read more in the MinION Mk1C IT requirements document.
Software for nanopore sequencing
MinKNOW
The MinKNOW software controls the nanopore sequencing device, collects sequencing data in real time and processes it into
basecalls. You will be using MinKNOW for every sequencing experiment. MinKNOW can also demultiplex reads by barcode, and
basecall/demultiplex data after a sequencing run has completed.
MinKNOW use
For instructions on how to run the MinKNOW software, please refer to the relevant section in the MinKNOW protocol.
EPI2ME (optional)
The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome,
barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-
basecalling.
EPI2ME installation and use
For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform
protocol.
Guppy (optional)
The Guppy command-line software can be used for basecalling and demultiplexing reads by barcode instead of MinKNOW. You can use
it if you would like to re-analyse old data, or integrate basecalling into your analysis pipeline.
Guppy installation and use
If you would like to use the Guppy software, please refer to the Guppy protocol.
Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be
done within three months of purchasing for MinION/GridION/PromethION flow cells, or within four weeks of purchasing for Flongle flow
cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result
is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the
flow cell check, please follow the instructions in the Flow Cell Check document.
Flow cell Minimum number of active pores covered by warranty
Flongle Flow Cell 50
MinION/GridION Flow 800

Cell
PromethION Flow Cell 5000
Library preparation
End-prep
~35 minutes
Materials 1 µg (or 100–200 fmol) amplicon DNA for every sample to be barcoded
Consumables 0.2 ml thin-walled PCR tubes

Nuclease-free water (e.g. ThermoFisher, AM9937)
NEBNext Ultra II End Repair / dA-tailing Module (NEB, E7546)
Agencourt AMPure XP beads
Freshly prepared 70% ethanol in nuclease-free water
Equipment P1000 pipette and tips

Thermal cycler at 20°C and 65°C
Microfuge
Hula mixer (gentle rotator mixer)
Magnetic rack
Ice bucket with ice
1 Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's
instructions, and place on ice:
For optimal perfomance, 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 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 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.
IMPORTANT
Do not vortex the NEBNext Ultra II End Prep Enzyme Mix.
IMPORTANT
It is important that the NEBNext Ultra II End Prep Reaction Buffer is mixed well by vortexing.
Check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise all precipitate.
2 Prepare the DNA in nuclease-free water.
Transfer 100–200 fmol amplicon DNA into a 1.5 ml Eppendorf DNA LoBind tube for R9.4.1 flow cells
Adjust the volume to 48 μl with nuclease-free water
Mix thoroughly by flicking the tube to avoid unwanted shearing
Spin down briefly in a microfuge
3 In a 0.2 ml thin-walled PCR tube, mix the following:
Reagent Volume
Amplicon DNA 48 µl
Ultra II End-prep reaction 3.5 µl

buffer
Ultra II End-prep enzyme mix 3 µl
Total 54.5 µl
4 Ensure the components are thoroughly mixed by pipetting, and spin down.
5 Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
IMPORTANT
AMPure XP bead clean-up
It is recommended that the repaired/end-prepped DNA sample is subjected to the following clean-up with AMPure XP beads. This
clean-up can be omitted for simplicity and to reduce library preparation time. However, it has been observed that omission of
this clean-up can: reduce subsequent adapter ligation efficiency, increase the prevalence of chimeric reads, and lead to an
increase in pores being unavailable for sequencing. If omitting the clean-up step, proceed to the next section.
6 Resuspend the AMPure XP beads by vortexing.
7 Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
8 Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube.
9 Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
10 Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
11 Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the
magnet, and pipette off the supernatant.
12 Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing
the pellet. Remove the ethanol using a pipette and discard.
13 Repeat the previous step.
14 Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds,
but do not dry the pellet to the point of cracking.
15 Remove the tube from the magnetic rack and resuspend the pellet in 25 µl nuclease-free water. Spin down and
incubate for 2 minutes at room temperature.
16 Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
17 Remove and retain 25 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µl of eluted sample using a Qubit fluorometer.
END OF STEP
Take forward the repaired and end-prepped DNA into the native barcode ligation step. However, at this point it is
also possible to store the sample at 4° C overnight.
Native barcode ligation

~50 minutes
Materials Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) if multiplexing more
than 12 samples
Consumables Freshly prepared 70% ethanol in nuclease-free water

Agencourt AMPure XP beads (Beckman Coulter™, cat # A63881)
NEB Blunt/TA Ligase Master Mix (NEB, M0367)
Equipment Magnetic separator, suitable for 1.5 ml Eppendorf tubes

Vortex mixer
Ice bucket with ice
Microfuge
Optional Qubit fluorometer (or equivalent for QC check)

Equipment
1 Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:
1. Thaw the reagents at room temperature.
2. Spin down the reagent tubes for 5 seconds.
3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
2 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.
3 Select a unique barcode for every sample to be run together on the same flow cell, from the provided 24 barcodes.
Up to 24 samples can be barcoded and combined in one experiment.
4 Dilute 100–200 fmol of each end-prepped sample to be barcoded to 22.5 µl in nuclease-free water.
5 Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
Reagent Volume
100–200 fmol end-prepped 22.5 µl

DNA
Native Barcode 2.5 µl
Blunt/TA Ligase Master Mix 25 µl
Total 50 µl
6 Mix well by pipetting using wide-bore pipette tips. Alternatively, if you are concerned about preserving the integrity
of very long DNA fragments, mix gently by flicking the tube, and spin down.
7 Incubate the reaction for 10 minutes at room temperature.
9 Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
11 Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
12 Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
13 Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing
the pellet. Remove the ethanol using a pipette and discard.
15 Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds,
but do not dry the pellet to the point of cracking.
16 Remove the tube from the magnetic rack and resuspend the pellet in 26 µl nuclease-free water. Incubate for 2
minutes at room temperature.
17 Pellet the beads on a magnet until the eluate is clear and colourless.
18 Remove and retain 26 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
Quantify 1 µl of eluted sample using a Qubit fluorometer.
IMPORTANT
Please first refer to the ligation step below to ensure that the library is diluted to the correct volume.
19 Pool equimolar amounts of each barcoded sample into a 1.5 ml Eppendorf DNA LoBind tube, ensuring that sufficient
sample is combined to produce a pooled sample of 100–200 fmol total.
20 Quantify 1 µl of pooled and barcoded DNA using a Qubit fluorometer.
21 Dilute 100–200 fmol pooled sample to 65 µl in nuclease-free water.
Optional Action
If 100–200 fmol for R9.4.1 of pooled sample exceeds 65 µl in volume, perform an AMPure clean-up with 2.5x Agencourt AMPure XP
beads to pooled sample volume, eluting in 65 µl of nuclease-free water.
Fragment size and adapter ligation
The amount of adapter has been optimised for fragment sizes greater or equal to 8 kb. If the fragments are generally smaller than 3
kb, adjustments should be made to use 0.1–0.2 pmoles of DNA in the adapter ligation step.
Adapter ligation and clean-up

~50 minutes
Materials Long Fragment Buffer (LFB)
Short Fragment Buffer (SFB)
Elution Buffer (EB) from the Oxford Nanopore sequencing kit
Adapter Mix II (AMII)
Consumables Quick T4 DNA Ligase in NEBNext® Quick Ligation Module (NEB, cat # E6056)
NEBNext® Quick Ligation Reaction Buffer (NEB, cat # B6058)
Agencourt AMPure XP beads
Equipment Microfuge
Magnetic rack
Vortex mixer
Optional Qubit fluorometer (or equivalent for QC check)

Equipment
Adapter Mix II Expansion use
Protocols that use the Native Barcoding Expansions require 5 μl of AMII per reaction. Native Barcoding Expansions EXP-
NBD104/NBD114 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 reactions when
sequencing on Flongle). This assumes that all barcodes are used in one sequencing run.
The Adapter Mix II expansion provides additional AMII for customers who are running subsets of barcodes, and allows a further 12
reactions (24 on Flongle).
1 Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:
1. Thaw the reagents at room temperature.
2. Spin down the reagent tubes for 5 seconds.
3. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
The NEBNext Quick Ligation Reaction Buffer (5x) 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 several seconds to
ensure the reagent is thoroughly mixed.
IMPORTANT
Do not vortex the Quick T4 DNA Ligase.
2 Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice.
3 Spin down the Adapter Mix II (AMII), pipette mix and place on ice.
IMPORTANT
Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either
enrich for DNA fragments of >3 kb, or purify all fragments equally.
To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB)
To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)
4 Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature, mix by vortexing,
spin down and place on ice.
5 Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each
sequential addition.
Reagent Volume
100–200 fmol for R9.4.1 pooled barcoded 65 µl

sample
Adapter Mix II (AMII) 5 µl
NEBNext Quick Ligation Reaction Buffer (5X) 20 µl
Quick T4 DNA Ligase 10 µl
Total 100 µl
6 Ensure the components are thoroughly mixed by pipetting, and spin down.
7 Incubate the reaction for 10 minutes at room temperature.
9 Add 50 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
11 Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
12 Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl 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.

14 Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30
seconds, but do not dry the pellet to the point of cracking.
15 Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB). Spin down and
incubate for 10 minutes at room temperature. For high molecular weight DNA, incubating at 37°C can improve the
recovery of long fragments.
16 Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
17 Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
18 Quantify 1 µl of adapter ligated DNA using a Qubit fluorometer - recovery aim 50–100 fmol.
IMPORTANT
We recommend loading 5-50 fmol of the final prepared library onto a flow cell.
Loading more than the maximal recommended amount of DNA can have a detrimental effect on output as higher quantities of
DNA results in a larger number of ligated DNA ends with loaded motor protein. This depletes fuel in the Sequencing Buffer,
regardless of whether or not the DNA fragments are being sequenced. This leads to fuel depletion and speed drop-off early in the
sequencing run. Dilute the libraries in Elution Buffer if required.
If you are using the Flongle for sample prep development, we recommend loading 3-20 fmol instead.
END OF STEP
The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.
TIP
Library storage recommendations
We recommend storing libraries in Eppendorf DNA LoBind tubes at4°C for short term storage or repeated use, for example,
reloading flow cells between washes.
For single use and long-term storage of more than 3 months, we recommend storing libraries at-80°C in Eppendorf DNA
LoBind tubes.
For further information, please refer to the DNA library stability Know-How document.
Optional Action
If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.
Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase
separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the
libraries onto flow cells.
Priming and loading the SpotON flow cell
~10 minutes
Materials Flow Cell Priming Kit (EXP-FLP002)

Loading Beads (LB)
Sequencing Buffer (SQB)
Consumables 1.5 ml Eppendorf DNA LoBind tubes

Equipment MinION Mk1B or Mk1C

SpotON Flow Cell
IMPORTANT
Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol.
TIP
Priming and loading a flow cell
We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.
1 Thaw the Sequencing Buffer (SQB), Loading Beads (LB), 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.
2 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.
3 Open the MinION device lid and slide the flow cell under the clip.
Press down firmly on the flow cell to ensure correct thermal and electrical contact.
1a
Insert the flow cell into the
device under the clip and
press down firmly.
Key:
Storage buffer
Priming mix
DNA library
1b
Insert the flow cell into the
device under the clip and
press down firmly.
Optional Action
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.
See the flow cell check instructions in the MinKNOW protocol for more information.
4 Slide the flow cell priming port cover clockwise to open the priming port.
IMPORTANT
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.
5 After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove
any bubbles:
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 ul, to draw back 20-30 ul, 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.
6 Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait
for 5 minutes. During this time, prepare the library for loading by following the steps below.
7 Thoroughly mix the contents of the Loading Beads (LB) by pipetting.
IMPORTANT
The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they
are mixed immediately before use.
8 In a new tube, prepare the library for loading as follows:
Reagent Volume per flow cell
Sequencing Buffer (SQB) 37.5 µl
Loading Beads (LB), mixed immediately before 25.5 µl

use
DNA library 12 µl
Total 75 µl
Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer (SQB) and Loading Beads (LB) because
the fuel in the buffer will start to be consumed by the adapter.
9 Complete the flow cell priming:
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.
5
Gently flip open the
SpotON
sample port cover.
10 Mix the prepared library gently by pipetting up and down just prior to loading.
11 Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each
drop flows into the port before adding the next.
12 Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port
and replace the MinION device lid.
9
Sequencing and data analysis
Data acquisition and basecalling
Overview of nanopore data analysis
For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the
Data Analysis document.
How to start sequencing
The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. It is assumed you
have already installed MinKNOW on your computer. There are multiple options for how to carry out sequencing:
1. Data acquisition and basecalling in real-time using MinKNOW on a computer
Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the
"Completing a MinKNOW run" section.
2. Data acquisition and basecalling in real-time using the GridION device
Follow the instructions in the GridION user manual.
3. Data acquisition and basecalling in real-time using the MinION Mk1C device
Follow the instructions in the MinION Mk1C user manual.
4. Data acquisition and basecalling in real-time using the PromethION device
Follow the instructions in the PromethION user manual or the PromethION 2 Solo user manual.
5. Data acquisition using MinKNOW on a computer and basecalling at a later time using MinKNOW or
Guppy
Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the
"Completing a MinKNOW run" section. When setting your experiment parameters, set the Basecalling tab to OFF. After the
sequencing experiment has completed, follow the instructions in the Post-run analysis section of the MinKNOW protocol or the Guppy
protocol starting from the "Quick Start Guide for Guppy" section.
Downstream analysis
Post-basecalling analysis
There are several options for further analysing your basecalled data:
1. EPI2ME platform
The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore
Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment,
and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret
report with the results. For instructions on how to run an analysis workflow in EPI2ME, please follow the instructions in the EPI2ME
protocol, beginning at the "Starting data analysis" step.
2. EPI2ME Labs tutorials and workflows
For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in
EPI2ME Labs, which are available in the EPI2ME Labs section of the Community. The platform provides a vehicle where workflows
deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code
and example data.
3. Research analysis tools
Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore
GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are
provided as-is, with minimal support.
4. Community-developed analysis tools
If a data analysis method for your research question is not provided in any of the resources above, please refer to theBioinformatics
section of the Resource centre. Numerous members of the Nanopore Community have developed their own tools and pipelines for
analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by
Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.
Ending the experiment
Materials Flow Cell Wash Kit (EXP-WSH004)
1 After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit
instructions and store the washed flow cell at 2-8°C, OR
The Flow Cell Wash Kit protocol is available on the Nanopore Community.
TIP
We recommend you to wash the flow cell as soon as possible after you stop the run. However, if this is not
possible, leave the flow cell on the device and wash it the next day.
2 Follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.
Instructions for returning flow cells can be foundhere.
All flow cells must be flushed with deionised water before returning the product.
IMPORTANT
If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting
Guide that can be found in the online version of this protocol.
Troubleshooting
Issues during DNA/RNA extraction and library
preparation
Below is a list of the most commonly encountered issues, with some suggested causes and solutions.
We also have an FAQ section available on theNanopore Community Support section.
If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email
([email protected]) or via LiveChat in the Nanopore Community.
Low sample quality
Observation Possible cause Comments and actions
Low DNA purity (Nanodrop reading The DNA extraction The effects of contaminants are shown in the Contaminants
for DNA OD 260/280 is <1.8 and OD method does not Know-how piece. Please try an alternative extraction method
260/230 is <2.0–2.2) provide the required that does not result in contaminant carryover.
purity
Consider performing an additional SPRI clean-up step.
Low RNA integrity (RNA integrity The RNA degraded Try a different RNA extraction method. For more info on RIN,
number <9.5 RIN, or the rRNA band during extraction please see the RNA Integrity Number Know-how piece.
is shown as a smear on the gel)
RNA has a shorter than expected The RNA degraded Try a different RNA extraction method. For more info on RIN,
fragment length during extraction please see the RNA Integrity Number Know-how piece.
We recommend working in an RNase-free environment, and to

keep your lab equipment RNase-free when working with RNA.
Low DNA recovery after AMPure bead clean-up

Low DNA loss due to a 1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to
recovery lower than intended the sample.
AMPure beads-to-
sample ratio 2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size
will be lost during the clean-up.
Low DNA fragments are The lower the AMPure beads-to-sample ratio, the more stringent the selection against short
recovery shorter than fragments. Please always determine the input DNA length on an agarose gel (or other gel
expected electrophoresis methods) and then calculate the appropriate amount of AMPure beads to
use.
Low The wash step used DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct
recovery ethanol <70% percentage.
after end-
prep
Issues during the sequencing run
Below is a list of the most commonly encountered issues, with some suggested causes and solutions.
We also have an FAQ section available on theNanopore Community Support section.
If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email
([email protected]) or via LiveChat in the Nanopore Community.
Fewer pores at the start of sequencing than after Flow Cell Check
Observation Possible Comments and actions
cause
MinKNOW An air bubble After the Flow Cell Check it is essential to remove any air bubbles near the priming port before
reported a was introduced priming the flow cell. If not removed, the air bubble can travel to the nanopore array and
lower number into the irreversibly damage the nanopores that have been exposed to air. The best practice to
of pores at the nanopore array prevent this from happening is demonstrated in this video.
start of
sequencing
than the
number
reported by
the Flow Cell
Check
MinKNOW The flow cell is Stop the sequencing run, remove the flow cell from the sequencing device and insert it again,
reported a not correctly checking that the flow cell is firmly seated in the device and that it has reached the target
lower number inserted into temperature. If applicable, try a different position on the device (GridION/PromethION).
of pores at the the device
start of
sequencing
than the
number
reported by
the Flow Cell
Check
MinKNOW Contaminations The pore count during the Flow Cell Check is performed using the QC DNA molecules present
reported a in the library in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate
lower number damaged or the number of active pores. Because of this, variability of about 10% in the number of pores is
of pores at the blocked the expected. A significantly lower pore count reported at the start of sequencing can be due to
start of pores contaminants in the library that have damaged the membranes or blocked the pores.
sequencing Alternative DNA/RNA extraction or purification methods may be needed to improve the purity
than the of the input material. The effects of contaminants are shown in the Contaminants Know-how
number piece. Please try an alternative extraction method that does not result in contaminant
reported by carryover.
the Flow Cell
Check
MinKNOW script failed

cause
MinKNOW shows Restart the computer and then restart MinKNOW. If the issue persists, please collect the
"Script failed" MinKNOW log files and contact Technical Support.
Pore occupancy below 40%
Pore Not enough library was loaded 5–50 fmol of good quality library can be loaded on to a MinION Mk1B/GridION flow
occupancy on the flow cell cell. Please quantify the library before loading and calculate mols using tools like
<40% the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore The Ligation Sequencing Kit Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford
occupancy was used, and sequencing Nanopore Technologies Ligation Buffer (LNB, provided in the SQK-LSK109 kit) at
close to 0 adapters did not ligate to the the sequencing adapter ligation step, and use the correct amount of each
DNA reagent. A Lambda control library can be prepared to test the integrity of the
third-party reagents.
Pore The Ligation Sequencing Kit Ethanol can denature the motor protein on the sequencing adapters. Make sure
occupancy was used, and ethanol was the LFB or SFB buffer was used after ligation of sequencing adapters.
close to 0 used instead of LFB or SFB at
the wash step after sequencing
adapter ligation
Pore No tether on the flow cell Tethers are adding during flow cell priming (FLT tube). Make sure FLT was added
occupancy to FB before priming.
close to 0
Shorter than expected read length
Shorter than Unwanted fragmentation Read length reflects input DNA fragment length. Input DNA can be
expected read length of DNA sample fragmented during extraction and library prep.
1. Please review the Extraction Methods in the Nanopore Community for

best practice for extraction.
2. Visualise the input DNA fragment length distribution on an agarose gel
before proceeding to the library prep.
In the image above, Sample 1 is of high molecular weight, whereas Sample

2 has been fragmented.
3. During library prep, avoid pipetting and vortexing when mixing reagents.
Flicking or inverting the tube is sufficient.
Large proportion of recovering pores

cause
cause
Large proportion of Contaminants Some contaminants can be cleared from the pores by the unblocking function
recovering pores (shown as are present in built into MinKNOW. If this is successful, the pore status will change to "single
dark blue in the channels the sample pores". If the portion of recovering pores (unavailable pores in the extended
panel and duty time plot) view) stays large or increases:
1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be
performed, or
2. Run several cycles of PCR to try and dilute any contaminants that may be
causing problems.
The duty time plot above shows an increasing proportion of "recovering" pores
over the course of a sequencing experiment
Large proportion of inactive pores

cause
Large proportion of inactive pores (shown as Air bubbles Air bubbles introduced through flow cell priming and
light blue in the channels panel and duty time have been library loading can irreversibly damage the pores. Watch
plot. Pores or membranes are irreversibly introduced into the Priming and loading your flow cell video for best
damaged) the flow cell practice
Large proportion of inactive pores Certain Known compounds, include polysaccharides, typically
compounds co- associate with plant genomic DNA.
purified with
DNA 1. Please refer to the Plant leaf DNA extraction method.
2. Clean-up using the QIAGEN PowerClean Pro kit.
3. Perform a whole genome amplification with the
original gDNA sample using the QIAGEN REPLI-g kit.
Large proportion of inactive pores Contaminants The effects of contaminants are shown in the
are present in Contaminants Know-how piece. Please try an alternative
the sample extraction method that does not result in contaminant
carryover.
Reduction in sequencing speed and q-score later into the run

Reduction in Fast fuel consumption is typically seen when the flow Add more fuel to the flow cell by following the
sequencing speed cell is overloaded with library (please see the instructions in the MinKNOW protocol. In future
and q-score later appropriate protocol for your DNA library to see the experiments, load lower amounts of library to the
into the run recommendation). flow cell.
Temperature fluctuation

cause
Temperature The flow cell Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the
fluctuation has lost contact flow cell and press it down to make sure the connector pins are firmly in contact with the device.
with the device If the problem persists, please contact Technical Services.
Failed to reach target temperature
MinKNOW The instrument was MinKNOW has a default timeframe for the flow cell to reach the target temperature.
shows placed in a location that Once the timeframe is exceeded, an error message will appear and the sequencing
"Failed to is colder than normal experiment will continue. However, sequencing at an incorrect temperature may lead
reach target room temperature, or a to a decrease in throughput and lower q-scores. Please adjust the location of the
temperature" location with poor sequencing device to ensure that it is placed at room temperature with good
ventilation (which leads ventilation, then re-start the process in MinKNOW. Please refer to this FAQ for more
to the flow cells information on MinION Mk 1B temperature control.
overheating)
Guppy – no input .fast5 was found or basecalled
No input .fast5 input_path did not point to The --input_path has to be followed by the full file path to the .fast5 files to be
was found or the .fast5 file location basecalled, and the location has to be accessible either locally or remotely
basecalled through SSH.
No input .fast5 The .fast5 files were in a To allow Guppy to look into subfolders, add the--recursive flag to the
was found or subfolder at the input_path command
basecalled location
Guppy – no Pass or Fail folders were generated after basecalling
No Pass or Fail The -- The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the
folders were qscore_filtering output folder, based on their strand q-score. When performing live basecalling in MinKNOW,
generated after flag was not a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads
basecalling included in the into Pass and Fail folders.
command
Guppy – unusually slow processing on a GPU computer
Unusually slow The --device flag The --device flag specifies a GPU device to use for accelerate basecalling. If not included in
processing on a wasn't included the command, GPU will not be used. GPUs are counted from zero. An example is --device
GPU computer in the command cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.
MinIT – the MinKNOW interface is not shown in the web browser
The MinKNOW Browser compatibility Always use Google Chrome as the browser to view MinKNOW. Alternatively,
interface is not issue instead of typing //mt-xxxxxx (x is a number) in the address bar, type in in the
shown in the web generic IP address, 10.42.0.1, which identifies the MinIT Wi-Fi router.
browser
The MinKNOW The MinIT Wi-Fi was not Make sure the computer or mobile device is using the MinIT Wi-Fi. It should be
interface is not used for connecting to the shown as MT-xxxxxx (x is a number) on the underside label on the MinIT:
shown in the web computer or mobile
browser device
Disable the Ethernet connection from the computer or mobile device as

needed. If necessary, contact your IT department to determine if the MinIT Wi-
Fi is blocked (MinIT generic IP: 10.42.0.1). Please white-list MinIT as needed.
The MinKNOW The MinIT was not on the Make sure that the wall sockets used by the Ethernet cables from the MinIT and
interface is not same network that the computer belong to the same local network.
shown in the web computer was connected
browser to.
MinIT – the MinIT software cannot be updated

cause
The MinIT The Please consult your IT department, as the MinIT software requires access to thefollowing AWS IP ranges.
software firewall Access to the following IP addresses is also needed:
cannot be is 178.79.175.200
updated blocking 96.126.99.215
IPs for
update
The MinIT The Occassionaly, the MinIT software admin page displays "updates available" even when the software is
software device already up-to-date. Please compare the version listed on the admin page with the one on the Software
cannot be already Downloads page. Alternatively, SSH into the MinIT through a SSH Client (e.g. Bitvise or Putty, as
updated has the described in the MinIT protocol) on a Windows computer or the terminal window on a Mac, run the
latest command, dpkg -l | grep minit, to find out the version of the MinIT software and sudo apt update if an
version update is needed. If the issue still persists, please contact Technical Services with details of the error.
of the
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Introduction to the protocol

Overview of the protocol

This is a Legacy product

Native Barcoding Expansion 1-12 and 13-24 features

These kits are recommended for users who:

wish to multiplex samples to reduce price per sample

Introduction to the Native Barcoding protocol

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

Prepare your library

You will need to:

150 min barcodes

You will need to:

Compatibility of this protocol

This protocol should only be used in combination with:

Ligation Sequencing Kit (SQK-LSK109)

Equipment and consumables

Consumables Agencourt AMPure XP beads (Beckman Coulter™, cat # A63881)

Equipment Hula mixer (gentle rotator mixer)

Optional Agilent Bioanalyzer (or equivalent)

How to QC your input DNA

Ligation Sequencing Kit contents (SQK-LSK109)

DNA CS DCS Yellow 1 50

Adapter Mix AMX Green 1 40

Ligation Buffer LNB Clear 1 200

Long Fragment Buffer LFB White cap, orange stripe on 2 1,800

Short Fragment Buffer SFB Grey 2 1,800

Sequencing Buffer SQB Red 2 300

Elution Buffer EB Black 1 200

Loading Beads LB Pink 1 360

Sequencing Tether SQT Purple 1 10

Flow Cell Priming Kit contents (EXP-FLP002)

Flush Buffer FB Blue 6 1,170

Flush Tether FLT Purple 1 200

Adapter Mix II Expansion contents (EXP-AMII001)

AMII : Adapter Mix II

Adapter Mix AMII Green 2 40

Adapter Mix II Expansion use

Native Barcoding Expansion 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114) contents

EXP-NBD104 kit contents

NBD NBD NBD NBD NBD NBD NBD

NBD NBD NBD NBD NBD

NBD01 : Native barcode 1 NBD08: Native barcode 8

Native Barcode 01-12 NB01-12 White 12 20

Adapter Mix II AMII Green 1 40

EXP-NBD114 kit contents

NBD NBD NBD NBD NBD NBD NBD

NBD NBD NBD NBD NBD

NBD13 : Native barcode 13 NBD20 : Native barcode 20

Native Barcode 13-24 NB13-24 White 12 20

Adapter Mix II AMII Green 1 40

Native barcode sequences

Component Forward sequence Reverse sequence

NB01 CACAAAGACACCGACAACTTTCTT AAGAAAGTTGTCGGTGTCTTTGTG

NB02 ACAGACGACTACAAACGGAATCGA TCGATTCCGTTTGTAGTCGTCTGT

NB03 CCTGGTAACTGGGACACAAGACTC GAGTCTTGTGTCCCAGTTACCAGG

NB04 TAGGGAAACACGATAGAATCCGAA TTCGGATTCTATCGTGTTTCCCTA

NB05 AAGGTTACACAAACCCTGGACAAG CTTGTCCAGGGTTTGTGTAACCTT