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Sequencing of client-made ONT libraries at the BRF

The ONT_Lib service offers sample QC, library preparation and sequencing of gDNA provided by clients. The workflow for this service is outlined below:

Sample submission

Please complete the ONT-Lib sample submission form in our online system BRF (IdeaElan – Facility : BRF Shared Equipment and Store). The sample submission form asks for:

• Research goal of your experiment
• Any QC data you have on your library (e.g. concentration readings and size traces).
• Acceptance of terms and conditions of service.

Once your libraries have been submitted electronically, bring them in person or by courier to the BRF. Ensure samples are:

• Labelled appropriately: Write the Sample Submission Id and the Request Id from the electronic form. Label cap and side of the tube.
• Transported in conditions that preserve sample integrity (e.g tubes are in good condition, properly sealed, transported cold, proper padding to avoid crushing, etc)

Initial quality control checks (Initial QC)

Once received, we will check your sample against the following:

Supported sample/library types²:

² R10.3 flow cells only support ligation kits (SQK-LSK109)

Project acceptance:

Upon QC completion, we will get in touch to discuss QC results and how to best proceed with sample processing. Your project will be marked as accepted when:

• Your libraries meet QC parameters as per table above.
• You have prepared your libraries using ONT kits and third party reagents supported reagents.
• There is an agreement between you and the BRF on what services will be included and their price. A quote for the service can be accepted electronically via IdeaElan.
• You have arranged enough electronic storage to transfer your data files to. We recommend ~1-2TB of disk per flow cell sequenced.

If libraries in your project do not meet QC parameters, you can:

• Withdraw the project: you will only be charged the for the QC fees. Reagents/consumables purchased for your project may also be charged at the BRF’s discretion.
• Resubmit your sample: you can reuse the sample submission form. QC for failed samples will still carry a fee.
• Proceed with the prep: Recommended only if it is apparent that your project goals can be met despite sample not meeting QC. The PI must provide written authorisation for the project to proceed.

Your library will be sequenced in the PromethION using ONT standard methods.

Data collection:

We will perform real-time base calling for each flow cell as per ONT recommendations. Customers can choose to receive fast5 or fast5 and fastQ files. Boutique basecalling/analysis methods are not offered as part of the standard service.

Important: ONT sequencing is a fast growing and developing technology, and as such, hardware/reagents/consumables occasionally may not perform as advertised. ONT is also extremely sensitive to the quality and purity of the input material you provide. At the BRF, we will strive to give you the best possible output and data quality as per your project goals, but due to the above mentioned limitations, we are unable to guarantee data outputs or read quality for your runs.

Data management and delivery

PromethION run outputs may exceed 1TB, which hinders out ability to provide long term data storage/back up for this service. To ensure a smooth and error-free data hand-over, we ask that data storage arrangements are made prior your sample is due to be sequenced.

• For clients with NCI accounts (ANU only): Transfer of data to an NCI account is the fastest and most reliable way to deliver data to you. If you are an ANU client, it is highly recommended you secure enough storage though an NCI project (nci.org.au). Please detail your NCI project and username in the electronic sample submission.
• For external clients: We will generate a transfer link using CloudStor. It is the client’s responsibility to download the data and check its integrity.
• If you work in collaboration with an ANU bioinformatics team (e.g. the ABC or EMBL Australia), you are encouraged to negotiate NCI storage through your collaborators.

We require acknowledgement that you have received your data within 5 working days after hand over. Data will be archived by the BRF for a maximum of 3 months, and a you will be charged a weekly storage fee until you confirm your data has been received successfully. We reserve the right to permanently delete data after we receive confirmation of reception and/or after 3 months from the date of data hand over.

Storing and backing up your data is solely your responsibility. We strongly advise you back up your data securely upon reception.