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 Flow Cell, generating a 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 in 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 ≤4Mb can expect to generate a minimum 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 and generating at least 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 to ensure it has enough pores for a good sequencing run.

Sample preparation

We have developed several optimised extraction methods to generate high quality genomic DNA from your 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. This method 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 gDNA extraction:

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

Automated library preparation, sequencing and analysis

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

Library preparation step	Process
Sample extraction
Bead binding	Addition of the DNA binding bead mix from the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit to your lysed sample(s)
Clean-up	gDNA purification and clean-up
Elution	Elution of your 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
Flow cell priming	Prime the flow cell for sequencing
Clean-up	Clean-up your samples by performing an Ethanol wash
Elution	Elute the pooled and cleaned DNA, and attach the sequencing adapters to the DNA ends
Flow cell loading	Prime the flow cell and load the prepared library for sequencing
Sequencing	Your sequencing run uses the Gourami 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 uing the isolate mode of the wf-bacterial-genomes workflow.
Flow cell wash or flush	Flow cell is washed using the EXP-WSH004 for reuse or flushed for return

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 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
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
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 10^8 – 10^9 cfu/ml) or 1/8 of a 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. Please 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 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.

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.

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

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 on the ElysION on-screen display.

Flow cell	Minimum number of active pores covered by warranty
MinION/GridION Flow Cell	800
PromethION Flow Cell	5000

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.

The sample sheet assigns a barcode to each sample, allowing a user 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 please refer to the ElysION 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 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

Note: The output 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.

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 7 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	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

Note: The output 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.

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 10^8 – 10^9 cfu/ml) or 1/8 of a 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. Please ensure you are following the correct method and using high-quality sample inputs.

Please 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 please refer to the ElysION User Guide.

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For commonly encountered issues please refer to the ElysION User Guide.

For in-depth troubleshooting please refer to the Set-up and operating manual: Early access ElysION provided with your ElysION device.

For additional customer support contact the nanoporetech support channels.