This end to end protocol describes our Nanopore-Only Microbial Isolate Sequencing Solution (NO-MISS): a flexible approach allowing sequencing of 4 to 24 microbial isolate genomes per MinION Flow Cell, generating a minimum coverage of 50x per genome.

The 50X coverage threshold is sufficient for downstream analyses including: accurate assembly and plasmid resolution, AMR profiling, core genome (cg) and whole genome (wg) multi-locus sequence typing (MLST), and cg/wgSNP typing. You can analyse your sequencing data using EPI2ME, which provides a user-friendly bioinformatics workflow.

We provide multiple DNA extraction approaches, depending on requirements, and starting organism (bacteria, fungi/yeast). These are key in achieving reliable flow cell output and genome coverage. The sample specific extraction methods use NEB Monarch® Spin gDNA Extraction Kit, while the universal method uses a bead-beating method and the Maxwell® RCS PureFood Pathogen Kit.

The extracted gDNA is then tagmented and sequenced using our Rapid Barcoding Kit (SQK-RBK114.24 or SQK-RBK114.96). Up to 24 samples per sequencing experiment for bacterial isolates (up to 7 Mb genomes) and up to 8 samples for fungi/yeast isolates can be processed to achieve the 50x coverage threshold. Use a minimum of four barcodes per run to maintain performance.
Detailed instructions for setting up the sequencing run on MinKNOW and downstream analysis are also included for a complete end-to-end protocol. We recommend sequencing up to 72 hours and generating at least 50x coverage per sample (approx. 0.5 Gb per barcode, assuming 5 Mb genome).

We recommend updating MinKNOW to the latest version prior to starting a sequencing run. The basecalling model v4.3 found in Dorado 0.5.0 onwards provides improved accuracy for bacterial DNA and is included in MinKNOW release v24.02 or newer.

For more information on updating MinKNOW, please refer to our MinKNOW protocol.

End-to-end workflow overview

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Extract your DNA, and check quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
• 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.

Sample preparation

Using the relevant gDNA extraction method, you will need to lyse your cells, extract your gDNA, and quantify the DNA:

• Universal bead-beating method:
• Universal bead-beating gDNA extraction:
  • For high throughput requirements and universal applications.

• Manual column-based methods:
• Bacteria gDNA extraction:
  • For bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Enterococcus faecalis, and Bacillus subtilis or staphylococci such as Staphylococcus aureus, and Staphylococcus epidermidis.
• Hard to lyse organisms gDNA extraction:
  • For bacteria, such as Mycobacterium tuberculosis.
• Fungi gDNA extraction:
  • For fungi/yeast, such as Candida albicans, Candida tropicalis, and Candida parapsilosis.

Library preparation

The table below is an overview of the steps required in the library preparation, including timings and stopping points.

Library preparation step	Process	Time	Stop option
DNA barcoding	Tagmentation of the DNA using the Rapid Barcoding Kit V14	15 minutes	4°C overnight
Sample pooling and clean-up	Pooling of barcoded libraries and AMPure XP Bead clean-up	25 minutes	4°C overnight
Adapter ligation	Attach the sequencing adapters to the DNA ends	5 minutes	We strongly recommend sequencing your library as soon as it is adapted
Priming and loading the flow cell	Prime the flow cell and load the prepared library for sequencing	5 minutes

Sequencing and analysis You will need to:

• Start a sequencing run using the MinKNOW software, which will collect raw data to basecall and demultiplex the barcoded reads.
• Perform downstream analysis uing the isolate mode of the wf-bacterial-genomes workflow in EPI2ME.

Input requirements per sample for the extraction methods:

• Universal bead-beating gDNA extraction: 1 ml liquid overnight culture (~1 x 10^8 – 10^9 cfu/ml) or half of a loop of colonies from a plate

• Bacteria gDNA extraction: 200 µl liquid overnight culture (~1 x 10^8 – 10^9 cfu/ml) or 1/8 of a loop of colonies from a plate

• Hard to lyse organisms gDNA extraction: 5 – 10 mg cells from solid or liquid media

• Fungi gDNA extraction: 2 ml of ~1 x 10^7 cfu/ml overnight culture or a full 10 µl inoculating loop from a plate

For library preparation, 200 ng in 10 µl of extracted gDNA per sample is required.

Depending on the extraction protocol used, not all third-party reagents are required.

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.

Custom CTAB buffer can be prepared instead of purchasing. Below are the reagents and concentrations required, with suggested examples with excess.

Reagent	Stock	Final concentration	Volume for 12 samples	Volume for 24 samples
CTAB	-	2% v/v	60 µl	120 µl
EDTA	0.5 M	40 mM	240 µl	480 µl
Sodium chloride	5 M	1.4 M	833 µl	1,666 µl
Trizma hydrochloride solution, pH 8	1 M	100 mM	300 µl	600 µl
Nuclease-free water	-	-	1567 µl	3,134 µl
Total	-	-	3,000 µl	6,000 µl

Staphylococcal Lysis Buffer (SLB) is required for the bacterial gDNA extraction method for staphylococcal inputs.

Reagent	Stock	Final concentration	Volume for 12 samples with excess	Volume for 24 samples with excess
Trizma hydrochloride solution, pH 9	1 M	100 mM	150 ul	300 µl
Sodium chloride	5 M	10 mM	3 ul	6 µl
SDS	10% v/v	0.1% v/v	15 ul	30 µl
Nuclease-free water	-	-	1332 ul	2664 µl
Total volume	-	-	1,500 ul	3,000 µl

The SDS in the Staphylococcal Lysis Buffer (SLB) is essential for preventing the degradation of staphylococci DNA. Exclusion of SDS from the buffer results in a larger smear of DNA when run on a gel.

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 the Input 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.

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 for MinION/GridION/PromethION or within four weeks of purchasing 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 Cell	800
PromethION Flow Cell	5000

We are in the process of reformatting the barcodes provided in this kit into a plate format. This will reduce plastic waste and facilitates automated applications.

Plate format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	1	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	2	1200
Elution Buffer	EB	Black	1	500
Sequencing Buffer	SB	Red	1	700
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	8000
Flow Cell Tether	FCT	Purple	1	200
Rapid Barcode plate	RB01-24	-	2 plates, 3 sets of barcodes per plate	5 µl per well

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.

Vial format

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	1	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	2	1,200
Elution Buffer	EB	Black	1	500
Sequencing Buffer	SB	Red	1	700
Library Beads	LIB	Pink	1	600
Library Solution	LIS	White cap, pink label	1	600
Flow Cell Flush	FCF	Blue	6	1,170
Flow Cell Tether	FCT	Purple	1	200
Rapid Barcodes	RB01-24	Clear	24	15

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.

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	2	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	3	1,200
Elution Buffer	EB	Black	1	1,500
Sequencing Buffer	SB	Red	1	1,700
Library Beads	LIB	Pink	1	1,800
Library Solution	LIS	White cap, pink label	1	1,800
Flow Cell Flush	FCF	Clear	1	15,500
Flow Cell Tether	FCT	Purple	2	200
Rapid Barcodes	RB01-96	-	3 plates	8 µl per well

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.

We have developed four optimised extraction methods to generate high quality genomic DNA from your cell cultures, allowing maximised sequencing output using this method.

What extraction method is right for me?

Sample extraction method	Sample type	Sample Input	Expected yield	Expected DNA Integrity Number (DIN)	Average sequencing read lengths	Extraction kits used
Universal bead-beating gDNA extraction	Universal applications: bacteria, fungi or yeast	1 ml liquid overnight culture (~1 x 10^8 – 10^9 cfu/ml) or half of a loop of colonies from a plate	>200 ng/µl per sample	7-9	~4-7 kb	QIAGEN PowerBead Tube and Promega Maxwell® RSC PureFood Pathogen kit
Bacteria gDNA extraction	Bacterial	200 µl liquid overnight culture (~1 x 10^8 – 10^9 cfu/ml) or 1/8 of a loop of colonies from a plate	~15-20 ng/µl per sample	9	>7 kb - Size will vary based on sample input species	NEB Monarch Spin gDNA Extraction Kit
Hard to lyse organisms gDNA extraction	Mycobacterium tuberculosis (or hard to extract bacterial samples)	5 – 10 mg cells from solid or liquid media	~15-40 ng/µl per sample	8	>7 kb - Size will vary based on sample input species	NEB Monarch Spin gDNA Extraction Kit
Fungi gDNA extraction	Fungi or yeast	2 ml of ~1 x 10^7 cfu/ml overnight culture or a full 10 µl inoculating loop from a plate	~40 ng/µl per sample	N/A	>7 kb - Size will vary based on sample input species	NEB Monarch Spin gDNA Extraction Kit

Note: The yield, DIN and sequencing read length of extracted DNA may vary depending on sample quality and species. Please ensure you are following the correct method and using high-quality sample inputs.

Follow the links in the table above for the extraction methods documentation.
Alternatively, these extraction methods can be found in the Extraction Protocols tab in the Documentation space on the Nanopore Community

This method has been developed to process 24 samples with a genome size of up to 7 Mb simultaneously.

For samples with a larger genome size ( >7 Mb), we recommend lowering the number of samples to be barcoded and sequenced simultaneously to 8 samples.

For optimal output, we currently do not recommend using fewer than 4 barcodes.

Note: This method provides a standardised process for sample throughput that we have validated in-house. These settings will be applicable to the majority of use-cases and we recommend all new users to follow the recommended method. Experienced users can adjust sample throughput (4 to 48 barcoded samples) based on sample quality, genome size, and coverage requirements.

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.

We recommend setting up a sequencing run on a MinION or GridION device using the basecalling and barcoding recommendations outlined below. All other parameters can be left to their default settings.

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a desktop application. EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis.

Further information about the available EPI2ME workflows are available here, along with the Quick Start Guide to start your first bioinformatic workflow.

For the accurate and reliable assembly or alignment of bacterial or fungal isolate genomes generated from the included protocols, we recommend using the wf-bacterial-genomes workflow. At its core, the workflow is an efficient assembly pipeline which also polishes genomes using Medaka.

Whilst running the workflow using the default parameters will produce high quality genome assemblies, using the ‘Isolates’ mode will perform additional analyses designed to increase genome quality and aid genome interrogation for common pathogens in the clinical and food safety fields. ‘Isolates’ analyses includes MLST(7-gene), species confirmation and AMR prediction in addition to sample-specific reports.

Note: You can also run this workflow through command line. However, we only recommend this option for experienced users. For more information, please visit the wf-bacterial-genomes page on GitHub.

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
Inefficient lysis	Enzyme activity has degraded in the solution or the isolate species is hard to lyse.	- Make a fresh enzyme solution. - Follow the hard to lyse gDNA extraction method. - Increase the enzyme incubation for longer than 10 minutes.
Low DNA concentration	Low input into the extraction method	- Check the cell input used - Add more input and perform the extraction again - Elute in less Elution Buffer - Concentrate the DNA with a 0.4X AMPure XP Bead wash.
Low DNA integrity number (DIN)	Low quality or concentration of sample input	- Repeat the extraction with freshly made enzyme solution - Concentrate the DNA input with a 0.4X wash
Low sequencing yield	Low sample concentration	- Concentrate the DNA with a 0.4X AMPure XP wash step to remove potential inhibitors. - Check the DNA concentration and quality. RNA presence may affect quantification of total DNA.
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 SPRI 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.
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 concentration of good quality library is loaded on to a MinION/GridION flow cell. To check the concentration, please refer to the library preparation protocol. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0	The Rapid Sequencing Kit V14/Rapid Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA	Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents.
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 Priming and loading your flow cell video for best practice
Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides, typically associate with plant genomic 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/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. Please refer to this FAQ for more information on MinION temperature control.