Rapid barcoding DNA V14 – automated ElysION (SQK-RBK114.96)

Version for device: MinION

1. Important This protocol is a work in progress and some details are expected to change over time. Please make sure you always use the most recent version of the protocol.
2. Introduction to the automated rapid barcoding DNA protocol using ElysION

   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 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 is able to generate high sequencing accuracies of over 99% (Q20+) and duplex data; but has been optimised for speed and simplicity. If users require high accuracy, we recommend using our Native Barcoding Kit 96 V14 kit (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 no clean-up steps) 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 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 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	Attach 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	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.

3. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • Rapid Barcoding Kit 96 V14 (SQK-RBK114.96)
   • R10.4.1 flow cells (FLO-MIN114)
   • Flow Cell Wash Kit (EXP-WSH004)
   • Flow Cell Priming Kit V14 (EXP-FLP004)
   • Sequencing Auxiliary Vials V14 (EXP-AUX003)
   • Rapid Adapter Auxiliary V14 (EXP-RAA114)
   • ElysION device

1. For this protocol, 330 ng gDNA in 30 µl is required per sample as input.

   The starting concentration of your sample(s) should be 11 ng/µl in 30 µl of volume.

   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.

2. Third-party reagents

   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.

3. 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.

4. 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

5. Important The Rapid Adapter (RA) used in this kit and protocol is not interchangeable with other sequencing adapters.
6. Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) contents

   

   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.

1. Sample sheet information

   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.

2. Set up your sample sheet as a .csv file with the following information:

   Required field	User input	Definition / field info
   v1		Version field, no input required outside of field
   assay	rbk:v1.0	Assay ID and version
   library_id	User-defined	Identification for the DNA library
   created_by	User-defined	Identification of operator setting up sample sheet
   created_at	User-defined	Date and time of creation. Please follow the format outlined in the ElysION user guide.
   sample_count	User-defined	Number of samples processed in run (1 to 96 samples)
   well_id	A1-H12	Positions in 96-well plate being used (1 to 96 samples). Samples must start from position A1 in the 96-well plate and run consecutively column-wise.
   barcode	barcode01-barcode96	Rapid barcodes from SQK-RBK114.96 being used in library preparation. Up to 96 barcodes are available in the sequencing kit, these should be used secuentially. (e.g. Barcodes 01-96, Barcodes 01-24, or Barcodes 50-60)
   sample_type	User-defined	Description or characteristics of sample input
   sample_id	User-defined from LIMS system (lims-sampleid-12345)	Identification for each sample input (e.g. from LIMS system)

   
   
   

   Below is an example of a sample sheet CSV file for 10 samples:

   

1. The automated sample extraction, library preparation and sequencing method using the ElysION device can be followed using the on-screen display on the device.

   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.

2. Thaw kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:

   Reagent	1. Thaw at room temperature	2. Briefly spin down	3. Mix well by pipetting
   Rapid Barcode Plate (RB01-96)	Not frozen	✓	✓
   Rapid Adapter (RA)	Not frozen	✓	✓
   AMPure XP Beads (AXP)	✓	✓	Mix by pipetting or vortexing immediately before use
   Elution Buffer (EB)	✓	✓	✓
   Adapter Buffer (ADB)	✓	✓	Mix by vortexing
   Flow Cell Flush (FCF)	✓	✓	✓
   Flow Cell Tether (FCT)	✓	✓	✓
   Sequencing Buffer (SB)	✓	✓	✓
   Library Beads (LIB)	✓	✓	Mix by pipetting or vortexing immediately before use
   Bovine Serum Albumin (BSA)	✓	✓	✓
   Wash Mix (WMX)	✓	✓	✓
   Wash Diluent (DIL)	✓	✓	Mix by vortexing
   Storage Buffer (S)	✓	✓	Mix by vortexing

3. Switch on the ElysION device and its computer following the user guide.
4. Important The Background Services application must run continuously whilst using the ElysION, and should not be closed.
5. Ensure the Background Services application is running.

   If the application is not running, double click the "Background Services" desktop application on your ElysION device.

6. Open the ElysION UI application.
7. Important Ensure the ElysION deck is clear of any labware before performing automated checks.

   Failure to ensure the deck is clear can lead to errors in checks or damage to the equipment.

8. Close the door to the robot and select "Initialise" on the display.

   Note: The ElysION device will perform automated checks.

   Allow the initialisation checks to complete before proceeding with the library preparation method.

9. Select "Run method" on the on-screen display.
10. Select the method “Rapid Barcoding Kit” on the on-screen display.

    Click next to proceed.

11. Insert a MinION and GridION Flow Cell into the MinION Mk1D device on the ElysION deck.

    Instructions for inserting a flow cell onto the ElysION device can be found in the ElysION User Guide.

12. Select "Check flow cell" on the on-screen display.

    Once flow cell check begins, click next to proceed.

13. Select "Import" on the on-screen display to upload your sample sheet.

    Once completed click "next" to proceed.

14. In a new 0.5 ml Sarstedt Screw tube, prepare the Rapid Adapter Master Mix as follows and pipette mix:

    Reagent	Volume
    Rapid Adapter (RA)	1.5 μl
    Adapter Buffer (ADB)	3.5 μl
    Total	5 μl

15. Prepare the remaining reagents in accordance with the reagent preparation page on the on-screen display.

    Once completed click "next" to proceed.

16. Once your flow cell check has successfully completed, set up your sequencing run conditions on the on-screen display:
    1. Select the sequencing time limit, or to stop run when the flow cell pores are depleted.
    2. Select a data target.
    3. Select whether to perform:
       3.1. A flow cell flush to return the used flow cell.
       3.2. A flow cell wash using the Flow cell wash kit (EXP-WSH004) to reuse your flow cell.

    Note: Reagent volumes and guidance for flow cell wash or flush will be given on the deck loading page. If you would like to perform a flow cell wash for re-use it is recommended to thaw the wash reagents at the same time as the library preparation reagents.

    For more information on the run conditions please refer to the ElysION User Guide.

17. Important Reagent and consumables requirements

    Please consider the following when preparing and loading your reagents and consumables into the ElysION device to mitigate risk of workflow failure and equipment damage:

    • Ensure that the correct consumables are used with each reagent.
    • Spin down all samples and reagents, making sure they do not contain bubbles.
    • Ensure plates are flat in their deck position and sit within the barriers on the deck, and tubes are fully inserted into the rack.
18. Set up the ElysION deck according to the deck layout outlined in the UI of on-screen display.

    Note: The position of the tip boxes and reagents will change depending on the sample count, and run setup. Please ensure you are following the instructions on the on-screen display correctly for your run.

19. Insert two empty waste bins into the disposable waste draw below the deck.
20. Close the door of the ElysION device.
21. Click "Begin run" to start the automated library preparation and sequencing.
22. End of Step Once the run is finished, unload labware, empty the bins, and discard or store in the freezer any unused reagents.

1. ElysION device troubleshooting

   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.