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 Genomic Purification 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.

Cómo realizar un control de calidad de la muestra inicial de ADN

Es importante que la muestra de ADN cumpla con los requisitos de cantidad y calidad. Usar demasiado ADN, poco o de mala calidad (p. ej., que esté muy fragmentado, que contenga ARN o contaminantes químicos), puede afectar a la preparación de la biblioteca.

Para realizar un control de calidad en la muestra de ADN, consulte el protocolo Input DNA/ RNA QC

Contaminantes químicos

Dependiendo de cómo se extraiga el ADN de la muestra cruda, ciertos contaminantes químicos pueden permanecer en el ADN purificado, lo cual afecta la eficacia de la preparación de la biblioteca y la calidad de la secuenciación. Encontrará más información sobre contaminantes en la página Contaminants de la comunidad Nanopore.

Antes de empezar el experimento de secuenciación, recomendamos verificar el número de poros disponibles, presentes en la celda de flujo. La comprobación deberá realizarse en los primeros tres meses desde su adquisición, si se trata de celdas de flujo MinION, GridION o PromethION, y en las primeras cuatro semanas tras la compra de celdas de flujo Flongle. Oxford Nanopore Technologies sustituirá cualquier celda de flujo con un número de poros inferior al indicado en la tabla siguiente, siempre y cuando el resultado se notifique dentro de los dos días siguientes a la comprobación y se hayan seguido las instrucciones de almacenamiento. Para verificar la celda de flujo, siga las instrucciones del documento Flow Cell Check.

Celda de flujo	Número mínimo de poros activos cubierto por la garantía
Flongle	50
MinION/GridION	800
PromethION	5000

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 Genomic DNA Purification 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 Genomic DNA Purification 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 Genomic DNA Purification 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.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.