The protocol includes two separate sequencing experiments, using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) and the Pore-C protocol with the Ligation Sequencing Kit V14 (SQK-LSK114). A total of three PromethION Flow Cells are recommended for telomere-to-telomere sequencing of a single human sample.

The protocol describes each experiment individually; however, the Ultra-Long DNA Sequencing Kit and Pore-C library preparation steps will be carried out across multiple days. All optional and required pause steps will be highlighted throughout the protocol.

This protocol was developed in collaboration with the UCSC Nanopore Production Center, led by Dr. Karen Miga.

For supplementary information on this end-to-end workflow please visit our know-how document: Telomere-to-telomere sequencing (T2T) know-how document.

To achieve in-depth telomere-to-telomere sequencing of a sample, two different datasets must be generated to give high-accuracy data, ultra-long reads, as well as chromatin conformation capture data. This protocol describes the generation of chromatin conformation capture data using the Pore-C protocol. It is also possible to use Hi-C data instead of Pore-C, and the analysis workflow includes both options.

The following two experiments are set up:

• Ultra-long DNA sequencing experiment: This experiment yields ultra-long reads using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114), generating a viscous library of ultra-long DNA fragments that requires careful handling to maintain the long fragments. The DNA extraction and library preparation processes are performed in duplicate side-by-side, taking ~3.5 hours with an overnight elution, yielding a total of ten library loads across two flow cells. This means five library loads are required per flow cell across a 140-hour sequencing run.

This experiment aims to generate 40–60x coverage of the human genome, with a read N50 of 60+ kb, although optimal assembly performance is observed with 50–60x coverage and a read N50 of 70–100kb (see the telomere-to-telomere sequencing (T2T) know-how document for further details on performance).

• Pore-C sequencing experiment: This experiment generates chromatin conformation capture data using the Pore-C protocol and the Ligation Sequencing Kit V14 (SQK-LSK114). The Pore-C DNA extraction takes ~3 hours of hands-on time over three days with two overnight steps. The library preparation step takes ~60 minutes of hands-on time and is loaded on one PromethION Flow Cell. This experiment has been developed by Oxford Nanopore Technologies and the following published literature: Lieberman-Aiden et al., 2009; Comet et al., 2011; Belton et al., 2012; Gavrilov, Golov and Razin, 2013; Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017; Ulahannan et al., 2019. This experiment intends to manipulate cell suspensions to capture three-dimensional interactions of DNA within chromatin. This workflow has been written using NlaIII restriction enzyme and the heat denaturation method. For further information on protocol considerations, please see the Restriction Enzyme Pore-C info sheet.

This protocol aims to generate 10x coverage Pore-C data.

Note that the Pore-C data must come from the same sample as the ultra-long read data.

Steps in the sequencing workflow

Prepare for your experiment

You will need to:

• Ensure you have your human cell line or fresh whole blood samples ready. Fresh blood samples which have not been frozen are essential.
• Ensure you have your sequencing kits, the correct equipment, and third-party reagents.
• Download the MinKNOW software for acquiring and analysing your data.
• Check your flow cells to ensure they have enough pores for a good sequencing run.

Protocol workflow

The Tables below are an overview of the steps required in each experiment, including timings and optional stopping points.

Ultra-long DNA experiment
Note: this experiment is performed in duplicate, each prep yielding five library loads per flow cell.

Steps	Process	Time	Stop option
Cell isolation	Isolate white blood cells from whole blood or cells from cell culture.	30 minutes	The custom SPRI beads can be made and stored at 4°C before use. 4°C overnight
Ultra-long DNA extraction	Extract ultra-long high molecular weight DNA.	190 minutes	Stored at room temperature overnight or store at 4°C for short-term storage.
Library preparation	Tagment your DNA using a diluted fragmentation mix, attach the sequencing adapters and clean up the sample by precipitating your DNA and eluting overnight.	190 minutes	Overnight elution at room temperature. 4°C short-term storage or for repeated use, such as reloading your flow cell. -80°C for single-use, long-term storage. We strongly recommend sequencing your library as soon as it is adapted. All excess adapted DNA library should be stored at 4°C or on ice until use.
Priming and loading your flow cell	Prime your flow cell and load the prepared library for sequencing.	~ 30 minutes (5 minutes hands-on time)
Washing and reloading your flow cell	Wash your flow cell and reload the prepared library for further sequencing every 20-24 hours until you have sequenced five library loads on a flow cell.	~ 45 minutes (5-10 minutes hands-on time)

Pore-C experiment

Steps	Process	Time	Stop option
Cell isolation	Isolate PBMC from whole blood or cells from cell culture.	30 minutes	The custom SPRI beads can be made and stored at 4°C before use. Snap freeze aliquots of white blood cells and store at -80°C until the experiment can begin.
Pore-C extraction	Crosslink the three-dimensional DNA interactions within the nucleus of isolated cells. Next, permeabilise the cells and denature the chromatin. Cleave the genome with a restriction enzyme and ligate the cohesive ends of proximal crosslinked monomers into chimeric Pore-C polymers held in proximity. Degrade the protein structures to release the chimeric Pore-C polymers into solution and finally, purify the Pore-C extract.	Day 1: 50 minutes hands on time, 2.5 hour procedure time and overnight Day 2: 10 minutes hands on time, 6 hours procedure time and overnight Day 3: 40 minute hands on time, 1 hour 50 procedure time and optional overnight step	Aside from the multiple overnight incubations, there are a couple of optional pause steps. Snap-freeze the crosslinked aliquots in liquid nitrogen. Store frozen sample pellets at –80°C and use within one year. The extracted DNA can be stored at 4°C overnight until library preparation.
Library preparation	Repair DNA and prepare the ends for sequencing adapter attachment.	60 minutes	Overnight storage at 4°C following DNA library elution. We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading your flow cell	Prime the flow cell and load the prepared library for sequencing.	~ 30 minutes (5 minutes hands-on time)

Sequencing and analysis

• For each experiment, start a sequencing run using the MinKNOW software which will collect raw data from the device. Live basecalling is performed to support output estimates during sequencing.

Further details for each experiment set-up and basecalling are outlined in the "Data acquisition and basecalling" section of each experiment.

• For the Ultra-long DNA experiment, re-basecall your data after sequencing has completed using Dorado.

• For the Pore-C experiment, proceed with the data that has been basecalled live in MinKNOW during sequencing.

Telomere-to-telomere assembly:

• Finally, perform the telomere-to-telomere assembly using your basecalled data from all three experiments following the "Downstream analysis" section of the protocol.

We recommend different sample preparations due to different input requirements for each experiment, with the option to use either human cell lines or human whole blood. Other methods are available and may be more appropriate for your lab; however, please ensure to yield enough input required for each library preparation. It is also worth noting that depending on how DNA is extracted from a 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.

Human whole blood: approximately a total of 10–15 ml of blood is required for all sample preparation steps. The whole blood can be collected in an anticoagulant such as K2-EDTA but we do not recommend mixing with other additives as they may interfere with the Pore-C DNA extraction or the DNA sequencing run. Fresh blood samples which have not been frozen are essential for Pore-C DNA extraction.

Human whole blood input requirements:

• Ultra-long DNA experiment: 3.2 ml
• Pore-C experiment: 5–10 ml

Human cell lines from culture can also be used. We recommend isolating DNA from cell culture using standard techniques. However, for the Assembly Polishing Kit experiment, we have included an extraction protocol from cell culture.

• Ultra-long DNA experiment: 6 million cells
• Pore-C experiment: 10 million cells

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

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 12 weeks of purchasing your PromethION Flow Cells. Oxford Nanopore Technologies will replace any unused flow cell with fewer than the number of pores listed 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
PromethION Flow Cell	5,000

Name	Acronym	Cap colour	Number of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	1	40
Fragmentation Mix	FRA	Amber	1	50
FRA Dilution Buffer	FDB	Clear	1	1,600
Elution Buffer	EB	Black	2	1,500
Extraction EB	EEB	Orange	3	1,700
Sequencing Buffer UL	SBU	Red	2	1,000
Loading Solution UL	LSU	White cap, pink label	1	200
Flush Tether UL	FTU	Purple	1	600
Flow Cell Flush	FCF	Blue	2	15,500
Precipitation Buffer	PTB	Blue	2	1,700
Precipitation Star	PS	Yellow	6	1 star

Name	Acronym	Cap colour	Number of vials	Fill volume per vial (µl)
Elution Buffer	EB	Black	1	1,500
Sequencing Buffer UL	SBU	Red	2	1,000
Loading Solution UL	LSU	White cap, pink label	1	200
Flush Tether UL	FTU	Purple	1	600
Flow Cell Flush	FCF	Clear cap, light blue label	1	15,500

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
DNA CS	DCS	Yellow	1	35
Ligation Adapter	LA	Green	1	40
AMPure XP Beads	AXP	Amber	1	1,200
Ligation Buffer	LNB	White	1	200
Long Fragment Buffer	LFB	Orange	2	1,800
Short Fragment Buffer	SFB	Clear	2	1,800
Sequencing Buffer	SB	Red	1	700
Elution Buffer	EB	Black	1	500
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Flow Cell Flush	FCF	Clear cap, light blue label	1	8,000
Flow Cell Tether	FCT	Purple	1	200

Note: This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Wash Mix	WMX	Brown	1	15
Wash Diluent	DIL	Brown	2	1,300
Storage Buffer	S	Brown	2	1,600

• Wash Mix (WMX) contains DNase I.
• Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
• The Storage Buffer allows flow cells to be stored for extended periods of time.

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Flow Cell Flush	FCF	Clear cap, light blue lable	6	8,000
Flow Cell Tether	FCT	Purple	1	200

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Elution Buffer	EB	Black	2	500
Sequencing Buffer	SB	Red	2	700
Library Solution	LIS	White cap, pink label	2	600
Library Beads	LIB	Pink	2	600
Flow Cell Flush	FCF	Clear cap, light blue label	2	8,000
Flow Cell Tether	FCT	Purple	2	200

Approximately 6 million isolated white blood cells must be prepared from 1.6 ml of whole blood to use as input in the ultra-long DNA experiment.

Users may isolate white blood cells by any means they think are most appropriate for the whole blood sample to be used. If an alternative method is used, this step can be skipped and proceed directly to the next section of the protocol.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate and we recommend performing this step for both volumes of blood side by side. Note: ensure both blood volumes are from the same sample.

In this step, ultra-long DNA is extracted from the isolated cells and quantified before proceeding to the library preparation step.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate and we recommend performing this step for both white blood cells isolated from whole blood side by side.

This method has been optimised using the Extraction EB (EEB) from the Oxford Nanopore sequencing kit.

In this step, the extracted ultra-long DNA is prepared for sequencing by tagmentation and rapid attachment of sequencing adapters.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates of 750 µl of extracted uHMW gDNA in EEB.

Once the ultra-long DNA library has been prepared, the PromethION Flow Cell can be primed, and the library prepared with the final sequencing reagents before loading for sequencing to begin. Due to the viscosity of the library, the flow cell priming and loading steps have been modified.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates.

We recommend reloading your PromethION Flow Cell with a fresh ultra-long DNA library to maintain high output, using the modified method for reloading a viscous library.

For the Ultra-long DNA experiment, up to five libraries prepared using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) can be loaded on the PromethION Flow Cell during a sequencing run. We recommend washing the flow cell when ~20-25% of active pores are remaining, which typically occurs after ~20-24 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare the flow cell for loading a new library for further sequencing.

Navigate to the pore activity or the pore scan results plot to see pore availability. Below is an example of pore states observed on a flow cell before and after wash steps are performed. The red asterisks indicates the reloads.

Due to the viscosity of the library, the flow cell washing and reloading steps have been modified. It is also recommended to remove the waste fluid before washing the flow cell and before reloading of an ultra-long DNA library after each priming step.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates.

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

We recommend first basecalling in real-time using the fast basecaller on MinKNOW using the PromethION 24 or 48 device. MinKNOW can be used and set up to sequence in multiple ways:

• On a computer either directly or remotely connected to a sequencing device.
• Directly on a PromethION 24/48 sequencing device or PromethION 2 Integrated sequencing device.

After real-time basecalling, re-basecall the data using the super-accurate (SUP) basecaller v5.0 model (or newer) in Dorado.

For more information on using MinKNOW on a sequencing device, please see the PromethION 24/48 user manual or PromethION 2 Integrated user manual.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

• PromethION 24 and 48: Starting a sequencing run with PromethION 24 and 48 PromethION 2 Solo: Starting a sequencing run on PromethION 2 Solo

To start a sequencing run on MinKNOW:

1. Navigate to the start page and click start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) on the Kit page.

4. Configure the sequencing parameters as follows:

Basecalling	OFF
Modified bases	OFF
Model	FAST basecalling
Barcoding	OFF [default]
Alignment	OFF [default] We do not currently recommend live alignment during sequencing, as it can slow down system processing.
Adaptive sampling	OFF [default]
Advanced options	default settings

5. Configure the data targets as follows:

Run duration	140 hours

6. Configure the analysis workflow as follows:

Workflow	OFF

7. Configure the output parameters as follows:

Basecalled output type	.BAM
Based on	Time elapsed
Frequency	Every 10 minutes
FASTQ options - Compression	ON [default]
Raw reads	ON [default]
POD5	ON [default]
FAST5	OFF [default]

8. Configure the filtering options as follows:

Filtering	ON [default]
Min Qscore	10 [default]
Min read length (kb)	1 kb

Once sequencing is complete, re-basecall your data using the super-accurate (SUP) basecaller using command line Dorado with the following commands as described on the Dorado Github page:

$ dorado basecaller --min-qscore 10 sup ultralong_pod5s/ > ulk_reads.bam

Note: when running Dorado, we recommend stopping other basecalling for the best performance by maximising available memory to Dorado. This can be stopped and re-started when Dorado has finished via the GUI on MinKNOW.

Please remember to change the output file name to differentiate between each of the ultra-long experiment flow cells run in duplicate.

In the Downstream analysis section, we outline further options for analysing your basecalled data for the telomere-to-telomore experiment.

Before starting the Pore-C experiment, a custom SPRI bead suspension needs to be prepared. This will be used to deplete non-chimeric monomers and to maximise the frequency of chimeric Pore-C polymers, improving purity ratios and read lengths before library preparation.

Before starting the Pore-C DNA extraction, the PBMCs must be isolated from whole blood whilst maintaining cell viability. Approximately 5–10 ml of whole blood should yield sufficient PBMCs for the Pore-C DNA extraction. If necessary, combine multiple aliquots of whole blood to achieve a final 5–10 ml pooled sample. Using the method below, approximately 10 million PBMCs are prepared in aliquots of 1x PBMCs supplemented with 2% FBS. Approximately 10 million PBMCs are taken forwards into the Pore-C sample preparation step.

We recommend using SepMate™ PBMC Isolation Tubes from STEMCELL Technologies. However, users may isolate PBMCs by any means they feel are most appropriate for the whole blood sample to be used, provided that:

• PBMCs are isolated as soon as possible from fresh blood and no later than 24 hours.
• PBMCs are isolated using a method optimised for cell viability.
• The whole blood is not mixed with any additives, except for anticoagulants (e.g. K2-EDTA), which are acceptable and will not interfere with the Pore-C DNA extraction.

The following reagents can be prepared in advance of the Pore-C experiment or at the start of day that they are needed.

Pore-C extraction is performed across three days to stabilise the three-dimensional interactions of the DNA in the nucleus before extracting the DNA.

50 minutes hands-on time, 2.5 hour procedure time + overnight

During day 1, the white blood cells are prepared for stabilising of the three-dimensional interactions of DNA in the nucleus by chemically cross- linking DNA and protein. The nuclei are then permeabilised to expose the crosslinked cytoskeleton cage and nuclear structures before the chromatin is denatured. The DNA is now accessible to the chosen restriction enzyme† which passively diffuses through the crosslinked cytoskeleton cage and nuclear structures to digest the genome at compatible recognition sites. The sample is incubated overnight which creates clusters of DNA fragments held in proximity by crosslinks between DNA and the cytoskeleton, preserving the original interactions which were crosslinked.

†This protocol has been written using NlaIII and the heat denaturation method as our investigations have found this 4-cutter is particularly suitable for Pore-C across many different species, yielding Pore-C extracts with high contact densities. For more information, please see the "Protocol considerations" section of our Restriction enzyme Pore-C info sheet.

10 minutes hands-on time, 6 hour procedure time + overnight

During day 2, the restriction enzymes are heat inactivated to prevent re-digesting ligated products. DNA ligase is added to the clusters of crosslinked DNA and passively diffuses through the crosslinked cytoskeleton cage to ligate the cohesive ends of proximal monomers into chimeric Pore-C polymers. After ligation, the ligated products can be released from the crosslinked cytoskeleton cages by an overnight proteinase K digestion. This releases the chimeric Pore-C polymers into solution as dsDNA.

40 minutes hands-on time, 110 minutes procedure time, optional overnight step.

During day 3, the chimeric Pore-C dsDNA polymers are purified from the solution of polypeptide fragments and residual reaction buffers. The peptides are removed by using a phenol:chloroform extraction, followed by an ethanol precipitation to purify the DNA from the residual reaction buffers and phenol. The final Pore-C DNA extract is a pool of chimeric dsDNA polymers made of multiple ligated monomers which are sequenced to determine DNA interactions, proximity in sequence space and the three-dimensional structures of chromatin within the nucleus.

In this step, the extracted Pore-C DNA is prepared for sequencing by repairing any nicks in the DNA and preparing the ends for sequencing adapter attachment.

Once the Pore-C DNA extracts have been prepared using the Ligation Sequencing Kit V14 (SQK-LSK114), the PromethION Flow Cell can be primed, and the library prepared with the final sequencing reagents for the first library load to be sequenced.

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

We recommend basecalling in real-time using the high-accuracy (HAC) basecaller on MinKNOW using the PromethION 24 or 48 device. MinKNOW can be used and set up to sequence in multiple ways:

• On a computer either directly or remotely connected to a sequencing device.
• Directly on a PromethION 24/48 sequencing device or PromethION 2 Integrated sequencing device.

For more information on using MinKNOW on a sequencing device, please see the PromethION 24/48 user manual or PromethION 2 Integrated user manual.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

• PromethION 24 and 48: Starting a sequencing run with PromethION 24 and 48 PromethION 2 Solo: Starting a sequencing run on PromethION 2 Solo

To start a sequencing run on MinKNOW :

1. Navigate to the start page and click start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Ligation Sequencing Kit V14 (SQK-LSK114) on the Kit page.

4. Configure the sequencing parameters as follows:

Basecalling	ON
Modified bases	OFF
Model	High-accuracy (HAC) basecalling
Barcoding	OFF [default]
Alignment	OFF [default] We do not currently recommend live alignment during sequencing, as it can slow down system processing.
Adaptive sampling	OFF [default]
Advanced options	default settings

5. Configure the data targets as follows:

Run duration	72 hours

6. Configure the analysis workflow as follows:

Workflow	OFF

7. Configure the output parameters as follows:

Basecalled output type	.BAM & .FASTQ
Based on	Time elapsed
Frequency	Every 10 minutes
FASTQ options - Compression	ON [default]
Raw reads	ON [default]
POD5	ON [default]
FAST5	OFF [default]

8. Configure the filtering options as follows:

Filtering	ON [default]
Min Qscore	9
Min read length (kb)	0.2 kb

Your Pore-C experiment data is basecalled live in MinKNOW during sequencing, using the high-accuracy (HAC) basecaller.

In the Downstream analysis section, we outline further options for analysing your basecalled data.

The sequencing device control, data acquisition, and real-time basecalling are carried out by the MinKNOW software.

We recommend basecalling with the high accuracy (HAC) basecaller in real-time with BAM selected as output type using the P2i or P24/P48 device.

You must generate a BAM file from your sequening run, as this is required for input into the wf-human-variation workflow.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

• PromethION 24 and 48: "Starting a sequencing run with PromethION 24 and 48"

• PromethION 2 Integrated user manual

Below are the recommended sequencing parameters for MinKNOW.

To start a sequencing run on MinKNOW for the long read de novo sequencing from blood using SQK-LSK114 – scalable (near) telomere-to-telomere method:

1. Navigate to the start page and click start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Ligation Sequencing Kit V14 (SQK-LSK114) on the Kit page.

4. Configure the sequencing parameters as follows:

Basecalling	ON [default]
Modified bases	ON with '5mC & 5hmC CG contexts' selected
Model	SUP basecalling
Barcoding	OFF [default]
Alignment	OFF [default] We do not currently recommend live alignment during sequencing, as it can slow down system processing.
Adaptive sampling	OFF [default]
Advanced options	Active channel selection: ON [default] Time between pore scans: 1.5 [default] Reserve pores: ON [default]

5. Configure the data targets as follows:

Run duration	100 hours

6. Configure the analysis workflow as follows:

Workflow	OFF

7. Configure the output parameters as follows:

Basecalled output type	.BAM
Based on	Time elapsed
Frequency	Every 10 minutes
FASTQ options - Compression	ON [default]
Raw reads	ON [default]
POD5	ON [default]
FAST5	OFF [default]

8. Configure the filtering options as follows:

Filtering	ON [default]
Min Qscore	10 [default]
Min read length	200 bp [default]

Due to the extended sequencing time, and the multiple flow cell washes and library reloads, we do not recommend re-using the flow cells used in this method.

Re-using these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Observation	Possible cause	Comments and actions
Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2)	The DNA extraction method does not provide the required purity	The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional AMPure bead clean-up step.

Observation	Possible cause	Comments and actions
Low recovery	DNA loss due to a lower than intended AMPure beads-to-sample ratio	1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample. 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 recovery	DNA fragments are shorter than expected	The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use.
Low recovery after end-prep	The wash step used ethanol <70%	DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct percentage.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Observation	Possible cause	Comments and actions
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	An air bubble was introduced into the nanopore array	After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video for how to load a PromethION Flow Cell.
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	The flow cell is not correctly inserted into the device	Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	Contaminations in the library damaged or blocked the pores	The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Observation	Possible cause	Comments and actions
MinKNOW shows "Script failed"		Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

Observation	Possible cause	Comments and actions
Pore occupancy <40%	Not enough library was loaded on the flow cell	Ensure the correct volume and concentration as stated on the appropriate protocol for your sequencing library is loaded onto the flow cell. Please quantify the library before loading and calculate fmols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to fmol".
Pore occupancy close to 0	The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA	Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the SQK-LSK114 kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0	The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation	Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0	No tether on the flow cell	Tethers are adding during flow cell priming (FCT tube). Make sure FCT was added to FCF before priming.

Observation	Possible cause	Comments and actions
Shorter than expected read length	Unwanted fragmentation of DNA sample	Read length reflects input DNA fragment length. Input DNA can be 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.

Observation	Possible cause	Comments and actions
Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot) The pore activity plot above shows an increasing proportion of "unavailable" pores over time.	Contaminants are present in the sample	Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores 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.

Observation	Possible cause	Comments and actions
Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged)	Air bubbles have been introduced into the flow cell	Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the how to load a PromethION Flow Cell video for best practice.
Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides. 1. Clean-up using the QIAGEN PowerClean Pro kit. 2. Perform a whole genome amplification with the original gDNA sample using the QIAGEN REPLI-g kit.
Large proportion of inactive/unavailable pores	Contaminants are present in the sample	The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Observation	Possible cause	Comments and actions
Temperature fluctuation	The flow cell has lost contact with the device	Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services.

Observation	Possible cause	Comments and actions
MinKNOW shows "Failed to reach target temperature"	The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating)	MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW.