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.

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

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.

本产品包含由贝克曼库尔特公司（Beckman Coulter, Inc）生产的 AMPure XP 试剂，并可与试剂盒一起于-20°C 下储存（试剂稳定性将不受损害）。

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.