The ElysION automated Native Barcoding Kit 96 V14 (SQK-NBD114.96) workflow is optimised for simplicity and speed to automatically prepare and sequence between 1 and 48 purified DNA input samples, then automatically wash the used flow cell for re-use. The workflow is PCR-free, removing the PCR bias and retains information about base modifications, which can be analysed using tools developed in the Nanopore Community.

Note: This kit has been optimised to generate long reads, high sequencing accuracies of over 99% (Q20+) and duplex data. However, in order for long reads to be observed in sequencing, long fragments need to be present in the initial sample input. Alternatively, if users require a faster speed to sequencing, we recommend using our Rapid Barcoding Kit 96 V14 kit (SQK-RBK114.96).

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.

Automated library preparation, sequencing and analysis

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

Library preparation step	Process
DNA repair and end-prep	Reparation the DNA, preparation the DNA ends for adapter attachment, and bead clean-up.
Native barcode ligation and clean-up	Attachment of the native barcodes to the DNA ends and bead clean-up.
Adapter ligation and clean-up	Attachment of the sequencing adapters to the DNA ends and bead clean-up.
Priming and loading the flow cell	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, we have a number downstream analysis options for your data available through EPI2ME.

For this protocol you will require the following sample input:

We strongly recommend loading your samples to a fixed molarity for the best experiment results:

• For 1-4 samples: 270 fmol DNA in 20 µl per sample
• For 5-48 samples: 110 fmol DNA in 20 µl per sample

However, if the molarity of your samples cannot be calculated please follow the recommendations below:

• For 1-4 samples: 1000 ng DNA in 20 µl per sample (50 ng/µl)
• For 5-48 samples: 200 ng DNA in 20 µl per sample (10 ng/µl)

Note: The output and, sequencing read length of extracted DNA may vary depending on sample quality and species. Please ensure you are using high-quality sample inputs.

Sample type method variables: gDNA or amplicon

This method is used for gDNA samples by default. There is an option to select amplicon sample input variable.

For more information on editing method configurator variables please visit the ElysION User Guide.

Amplicon samples do not require DNA end repair, and therefore the End-Prep Master Mix formulation will differ depending on your sample type. Please follow the instructions on the on-screen display to ensure you are using the correct method for your run settings.

Third-party reagents

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.

Input DNA

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.

Check your flow cell

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)
Native Barcode plate	NB01-96	-	3 plates	8 µl per well
DNA Control Sample	DCS	Yellow	3	35
Native Adapter	NA	Green	2	40
Sequencing Buffer	SB	Red	2	700
Library Beads	LIB	Pink	2	600
Library Solution	LIS	White cap, pink label	2	600
Elution Buffer	EB	Black	1	1,500
AMPure XP Beads	AXP	Amber	1	6,000
Long Fragment Buffer	LFB	Orange	1	7,500
Short Fragment Buffer	SFB	Clear	1	7,500
EDTA	EDTA	Clear	1	700
Flow Cell Flush	FCF	Blue	1	15,500
Flow Cell Tether	FCT	Purple	2	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.

The barcodes are orientated in columns in the barcode plate.

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

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.

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.

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.