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

The 50x coverage threshold is sufficient for downstream analysis including accurate assembly and plasmid resolution, AMR profiling, core genome (cg) and whole genome (wg) multi-locus sequence typing (MLST), and cg/wgSNP typing. Your sequencing data will be analysed by the device using the wf-bacterial-genomes workflow, which produces a user-friendly report of the results.

We provide multiple DNA extraction approaches, depending on requirements, and starting organism (bacteria, fungi/yeast). These are key to achieving reliable flow cell output and genome coverage.

The extracted gDNA is then tagmented and sequenced using our Rapid Barcoding Kit (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. Bacteria with a genome size of ≤4 Mb can be expected to generate a coverage of 50x per genome. Please note, coverage may vary depending on larger genome size and input sample quality.

We recommend sequencing up to 72 hours or running to generate 50x coverage per sample (approx. 0.5 Gb per barcode, assuming 5 Mb genome).

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Ensure your ElysION device is installed with the required hardware and software package for this workflow.
• Ensure you have your sequencing kit, the correct equipment and third-party reagents.
• Check your flow cell during the ElysION run setup to ensure it has sufficient pores for a good sequencing run.

Sample preparation

We have developed several optimised extraction methods to generate high quality genomic DNA from cell cultures, allowing maximised sequencing output using the automated Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) end-to-end protocol.

What extraction method is right for me?

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

• Universal bead-beating method:

  • For high throughput requirements and universal applications, including bacteria and fungi.

• Bacteria gDNA extraction:

  • This extraction method is recommended for use with general bacterial samples. It has been validated with 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 for gDNA extraction:

  • This extraction method is recommended for hard to lyse organisms, such as Mycobacterium tuberculosis, or for bacteria that was not successfully extracted using the Bacterial gDNA extraction method.

Automated library preparation, sequencing, and analysis

The Table below is an overview of the steps automated by the ElysION device.

Steps	Process
Sample extraction
Bead binding	Addition of the DNA binding bead mix from the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit to the lysed sample(s)
Clean-up	gDNA purification and clean-up
Elution	Elution of the extracted and cleaned sample gDNA
Library preparation
DNA barcoding	Tagmentation of the DNA using the Rapid Barcoding Kit V14
Sample pooling	Pooling of barcoded samples
Bead binding	Addition of AMPure XP Beads
Clean-up	Clean-up of samples using an ethanol wash
Elution	Elution of pooled and cleaned DNA, and attachment of sequencing adapters to the DNA ends.
Priming and loading the flow cell	Prime the flow cell and load the prepared library for sequencing.
Sequencing	The sequencing run uses the MinKNOW software, which will collect raw data to basecall and demultiplex the barcoded reads.
Data analysis	Once sequencing is complete, the device will perform downstream analysis of the data using the wf-bacterial-genomes workflow.
Flow cell wash or flush	Wash the flow cell using the Flow Cell Wash Kit (EXP-WSH004) for reuse or flush the flow cell for return to Oxford Nanopore for recycling.

Sample extraction method	Sample type	Sample input	Expected yield	Expected DNA Integrity Number (DIN)	Average sequencing read lengths
Universal bead-beating gDNA extraction	Universal applications: bacteria, fungi or yeast	1 ml liquid overnight culture (~1 x 108 – 109 cfu/ml) or Half of a 10 µl loop of colonies from a plate	>200 ng/µl per sample	7-9	~4-7 kb
Bacteria gDNA extraction	Bacteria	200 µl liquid overnight culture (~1 x 108 – 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate	15-20 ng/µl per sample	9	>7 kb - Size will vary based on sample input species
Hard to lyse organisms gDNA extraction	Hard to extract bacterial samples (e.g. Mycobacterium tuberculosis)	For hard to lyse bacterial samples: 200 µl liquid overnight culture (~1 x 108 – 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate For Mycobacterium tuberculosis: 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

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

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 µl	300 µl
Sodium chloride	5 M	10 mM	3 µl	6 µl
SDS	10% v/v	0.1% v/v	15 µl	30 µl
Nuclease-free water	-	-	1,332 µl	2,664 µl
Total volume	-	-	1,500 µl	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.

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.

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

The number of pores in your flow cell will be checked by the ElysION device at the start of your assay. MinION/GridION Flow Cells should be checked within 12 weeks of purchasing.

Oxford Nanopore 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, follow the instructions on the ElysION on-screen display.

Flow cell	Minimum number of active pores covered by warranty
MinION/GridION Flow Cell	800

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

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.

The sample sheet assigns a barcode to each sample, allowing you to pick a range from the 96-well plate, and for a sample ID to be tracked from input to results.

For more information on setting up a sample sheet, refer to the ElysION MinION user guide.

This extraction method is a universal gDNA extraction from bacteria and fungi/yeast. It uses vortex bead-beating followed by a proteinase K and RNase treatment.

Note: If your desired yield is not achieved or you are looking to maximise the contiguity of your assemblies by generating longer read lengths, try one of our sample-tailored extraction methods:

• Bacteria gDNA extraction
• Hard to lyse organisms gDNA extraction

Sample extraction method	Sample type	Sample input	Expected yield	Expected DNA Integrity Number (DIN)	Average sequencing read lengths
Universal bead-beating gDNA extraction	Universal applications: bacteria, fungi or yeast	1 ml liquid overnight culture (~1 x 108 – 109 cfu/ml) or half of a 10 µl loop of colonies from a plate	>200 ng/µl per sample	7-9	~4-7 kb

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

This extraction protocol is recommended for use with general bacterial samples. We have validated this method with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Enterococcus faecalis, and Bacillus subtilis or staphylococci such as Staphylococcus aureus, and Staphylococcus epidermidis.

If the desired yield is not achieved from your extraction, try one of the following recommendations:

• The enzymatic reaction time in step 6 can be increased.
• Use the staphylococcal enzyme.
• Follow the Hard to lyse organisms gDNA extraction method.

Sample extraction method	Sample type	Sample input	Expected yield	Expected DNA Integrity Number (DIN)	Average sequencing read lengths
Bacteria gDNA extraction	Bacteria	200 µl liquid overnight culture (~1 x 108 – 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate	15-20 ng/µl per sample	9	>7 kb - Size will vary based on sample input species

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

This extraction method is recommended for hard to lyse organisms, such as Mycobacterium tuberculosis, or for bacteria that was not successfully extracted using the Bacteria gDNA extraction method.

Sample extraction method	Sample type	Sample input	Expected yield	Expected DNA Integrity Number (DIN)	Average sequencing read lengths
Hard to lyse organisms gDNA extraction	Hard to extract bacterial samples (e.g. Mycobacterium tuberculosis)	For hard to lyse bacterial samples: 200 µl liquid overnight culture (~1 x 108 – 109 cfu/ml) or 1/8 of a 10 µl loop of colonies from a plate For Mycobacterium tuberculosis: 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

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

Ensure you have all the correct hardware components, software packages and workflows installed to carry out this method.

For more information on the device and the processes, refer to the ElysION MinION user guide.

Method variables

Refer to the ElysION MinION user guide for more details.

If you wish to alter the default variables for your assay, select the System tab on the bottom left of the screen, then Method configurator. Then select the method that you wish to modify.

The variables for the NO-MISS protocol are detailed below:

Variable name	Default parameter	Parameter range	Description
Extraction Bead Binding Time (unit = seconds)	5 s	Min = 5 s Max = 1,800 s	Allows you to adjust the bead binding incubation time during extraction.
Extraction Bead Elution Time (unit = minutes)	5 min	Min = 2 min Max = 30 min	Allows you to adjust the extraction bead elution time.
Extraction Bead Pelleting Time (unit = seconds)	300 s	Min = 120 s Max = 1,800 s	Allows you to adjust the extraction bead pelleting time.
Pause for QC post Extraction	0 = off	0 = off 1 = on	Enabling this feature will instruct the ElysION to pause post extraction, allowing you to QC the samples.
AMPure XP Bead Clean-up Ratio	1.0X	0.4X, 0.6X, 0.8X, 1.0X, 1.2X	Allows you to adjust the ratio of AXP bead volume to sample volume during the clean-up step. For example, for the default value of 1.0, 100 μl of beads will be added to 100 μl of pooled samples. For a value of 0.6, 60 μl of beads will be added to 100 μl of pooled samples.
Library Prep Bead Binding Time (unit = minutes)	5 min	Min = 2 min Max = 30 min	Allows you to adjust the library prep bead binding time.
Library Prep Bead Elution Time (unit = minutes)	15 min	Min = 2 min Max = 30 min	Allows you to adjust the library prep bead elution time.
Pause for QC pre Flow Cell Loading	0 = off	0 = off 1 = on	Enabling this feature will instruct the ElysION to pause prior to the final eluate collection, allowing sample normalisation to a desired concentration, before automated flow cell loading.

Recommendations for the "Pause for QC" variable when enabled are provided at the end of this section.

Pause for QC post Extraction

Pause for QC pre Flow Cell Loading

Interleaving run setup

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, 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 option. - 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.
Low DNA integrity number (DIN)	Low quality or concentration of sample input	Repeat the extraction with freshly made enzyme solution.
Low sequencing yield	Low sample concentration	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. Try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step.

For commonly encountered issues, refer to the ElysION MinION user guide.

For in-depth troubleshooting, refer to the ElysION Operating manual provided with your device.

For additional customer support, contact the Oxford Nanopore support channel (support@nanoporetech.com).