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 to 48 purified DNA samples. This protocol is compatible with both genomic and amplicon DNA as input. 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 and high sequencing accuracies of over 99% (Q20+). However, in order for long reads to be observed in sequencing, long fragments need to be present in the initial sample input. Alternatively, if you require a faster turnaround time, 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 during the ElysION run setup to ensure it has sufficient 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	DNA repair, preparation of 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	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, there are a number of downstream analysis options for your data, available through EPI2ME.
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 type method variables: gDNA or amplicon

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

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 Native Barcoding protocol are detailed below:

Variable name	Default parameter	Parameter range/options	Description
gDNA or Amplicon Preparation	0 = gDNA	0 = gDNA 1 = Amplicon	Allows you to select the sample type for the prep.
End-Repair 20°C Incubation Time (unit = seconds)	300 s	Min = 120 s Max = 1,800 s	Allows you to adjust the length of the 20°C hold on the On-Deck Thermal Cycler (ODTC) during the end-repair reaction.
End-Repair 65°C Incubation Time (unit = seconds)	300 s	Min = 120 s Max = 1,800 s	Allows you to adjust the length of the 65°C hold on the ODTC during the end-repair reaction.
End-Repair AMPure XP Bead Clean-up Ratio	1.0X	0.4X, 0.6X, 0.8X, 1.0X, 1.2X, 1.4X, 1.6X, 1.8X, 2.0X, 2.2X, 2.4X, 2.6X, 2.8X, 3.0X	Allows you to adjust the ratio of AXP bead volume to sample volume during the end-repair clean-up step. For example, for the default value of 1.0, 100 µl of beads will be added to 100 µl of sample. For a value of 0.6, 60 µl of beads will be added to 100 µl of sample.
End-Repair Bead Binding Time (unit = seconds)	300 s	Min = 120 s Max = 1,800 s	Allows you to adjust the incubation time after the AXP bead addition, during the end-repair clean-up.
End-Repair Elution Time (unit = seconds)	120 s	Min = 120 s Max = 1,800 s	Allows you to adjust the length of time the beads are left to elute before pelleting, during the end-repair clean-up.
Pause for QC post End-Repair	0 = off	0 = off 1 = on	Enabling this feature will instruct the ElysION to pause post end-repair, allowing you to QC the samples.
Barcode Ligation Incubation Time (unit = seconds)	1,200 s	Min = 600 s Max = 3,600 s	Allows you to adjust the duration of the native barcode ligation step.
Barcode Ligation AMPure XP Bead Clean-up Ratio	0.4X	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 barcode ligation clean-up.
Barcode Ligation Bead Binding Time (unit = minutes)	10 min	Min = 2 min Max = 30 min	Allows you to adjust the incubation time after the AXP bead addition, during the barcode ligation clean-up.
Barcode Ligation Elution Time (units = minutes)	10 min	Min = 2 min Max = 30 min	Allows you to adjust the length of time the beads are left to elute before pelleting, during the barcode ligation clean-up.
Pause for QC post Barcode Ligation	0 = off	0 = off 1 = on	Enabling this feature will instruct the ElysION to pause post barcoding, allowing normalisation of samples.
Adapter Ligation Time (unit = seconds)	1,200 s	Min = 600 s Max = 3,600 s	Allows you to adjust the duration of the adapter ligation step.
Adapter Ligation AMPure XP Bead Clean-up Ratio	0.4X	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 adapter ligation clean-up.
Adapter Ligation Bead Binding Time (unit = minutes)	10 min	Min = 2 min Max = 30 min	Allows you to adjust the incubation time after the AXP bead addition, during the adapter ligation clean-up.
Adapter Ligation Wash Buffer	0 = LFB	0 = LFB 1 = SFB	Allows you to select either LFB or SFB to be used in the washing steps during the adapter ligation clean-up.
Adapter Ligation Elution Time (unit = minutes)	15 min	Min = 2 min Max = 30 min	Allows you to adjust the length of time the beads are left to elute before pelleting, during the adapter ligation clean-up.
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.

Notes:

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

• Recommendations for the "Pause for QC" variable when enabled are provided at the end of section 4, "ElysION run setup".

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, 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. 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

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.

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

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	Clear cap, light teal label	1	6,000
Long Fragment Buffer	LFB	Clear cap, orange label	1	7,500
Short Fragment Buffer	SFB	Clear	1	25,000
EDTA	EDTA	Blue	1	700
Flow Cell Flush	FCF	Clear cap, light blue label	1	15,500
Flow Cell Tether	FCT	Purple	2	200

Note:

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

• 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 oriented in columns in the barcode plate.

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.

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.

Pause for QC post End-Repair

Pause for QC post Barcode Ligation

Pause for QC pre Flow Cell Loading

Interleaving run setup

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).