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.

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.

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.

Cómo realizar un control de calidad del ADN de la muestra inicial

Es importante que la muestra de ADN cumpla con los requisitos de cantidad y calidad. Usar demasiado ADN, poco o de mala calidad (p. ej., que esté muy fragmentado, que contenga ARN o contaminantes químicos), puede afectar a la preparación de la biblioteca.

Para realizar un control de calidad en la muestra de ADN, consulte el protocolo Input DNA/ RNA QC

Contaminantes químicos

Dependiendo de cómo se extraiga el ADN de la muestra cruda, ciertos contaminantes químicos pueden permanecer en el ADN purificado, lo cual afecta la eficacia de la preparación de la biblioteca y la calidad de la secuenciación. Encontrará más información sobre contaminantes en la página Contaminants de la comunidad Nanopore.

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.

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.

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.