The ElysION automated Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) workflow is optimised for simplicity and speed to automatically prepare and sequence between 1 and 96 purified DNA samples. 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. This protocol is compatible with both genomic and amplicon DNA as input. If PCR amplicons are used, no base modifications will be retained.

Note: This kit is able to generate high sequencing accuracies of over 99% (Q20+) but has been optimised for speed and simplicity with fragmented DNA. If you wish to maintain DNA input length for optimal assembly and modifications, we recommend using our Native Barcoding Kit 96 V14 (SQK-NBD114.96).

Due to the simple nature of the workflow and the fact that little sample manipulation is required (e.g. minimal pipetting steps and one clean-up step), long reads can be achieved with this kit, despite the required transposase fragmentation. However, in order for long reads to be observed in sequencing, long fragments need to be present in the initial sample input.

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 barcoding	Tagmentation of the DNA using the Rapid Barcoding Kit V14.
Sample pooling and clean-up	Pooling of barcoded libraries and AMPure XP Bead clean-up.
Adapter ligation	Attachment of the 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 (optional)	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 Rapid 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.
Fragmentation Time - Step 1 (unit = seconds)	120 s	Min = 60 s Max = 240 s	Allows you to adjust the length of the 30°C hold on the On-Deck Thermal Cycler (ODTC) during the fragmentation reaction.
Fragmentation Time - Step 2 (unit = seconds)	120 s	Min = 60 s Max = 240 s	Allows you to adjust the length of the 80°C hold on the ODTC during the fragmentation reaction.
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.
Bead Binding Time (unit = minutes)	5 min	Min = 2 min Max = 30 min	Allows you to adjust the length of time the AXP-pool mix is incubated on the shaker.
Bead 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.
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.

Recommendation for the "Pause for QC" variable when enabled is provided at the end of section 4, "ElysION run setup".

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.

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

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)
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 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 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).