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Introduction to the protocol
This protocol outlines three methods for recovering a library for either transfer to a new flow cell or to wash the original flow cell for further sequencing of the recovered library. We recommend using these protocols to maximise sequencing output per sample or to provide additional support where a flow cell has failed mid-run.
A DNA library generated using any of our sequencing kits can be recovered and transferred for both MinION and PromethION Flow Cells. We recommend that a library can be recovered up to 2-3 times for successful sequencing on either a washed or a new flow cell.
Steps in the workflow:
Prepare for your experiment
You will need to:
- If using a new flow cell, check to ensure it has enough pores for a good sequencing run
- Ensure you have enough flow cell priming reagents, the correct equipment and third-party reagents
Method 1
Transfer a library between flow cellsYou will need to:
- Stop sequencing on MinKNOW
- Prime the second flow cell
- Recover the library from the original flow cell
- Transfer to the new flow cell within two hours
- Start a new sequencing run on MinKNOW
Method 2
Clean up and transfer a library between flow cellsYou will need to:
- Stop sequencing on MinKNOW
- Recover the library from the original flow cell
- SPRI clean the recovered library
- Store the library
- Prime the second flow cell
- Transfer to the new flow cell
- Start a new sequencing run on MinKNOW
Method 3
Recover a library to replace on a washed flow cellYou will need to:
- Pause sequencing on MinKNOW
- Recover the library from the original flow cell
- Flush the flow cell with flow cell Wash Mix
- Reprime the washed flow cell
- Reload the library onto the washed flow cell
- Resume the sequencing run on MinKNOW
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Use cases for each method
Method 1: Transfer a library between flow cells
- For users wanting to continue generating data from the same library after a sequencing run has completed.
- If a flow cell has failed within a few hours of sequencing, the library can be recovered and loaded onto a new flow cell to continue sequencing.
Method 2: Clean up and transfer a library between flow cells- For users wanting to continue generating data from the same library at a later date after a sequencing run has completed or if a second flow cell is not immediately available for transfer.
- If the flow cell was not prepared with the compatible flow cell reagents for the library chemistry, the library can be cleaned up and transferred to a flow cell primed with the correct buffers.
- For example, if a Kit 14 DNA library is loaded onto an R10.4.1 flow cell primed with Kit 9 flow cell priming reagents, the DNA library should be recovered, SPRI cleaned and loaded onto a flow cell primed with Kit 14 compatible reagents.
- If a flow cell has failed within a few hours of sequencing, the library can be recovered and loaded onto a new flow cell to continue sequencing at a later date.
Method 3: Recover a library to replace on a washed flow cell- For users wanting to continue generating data from the same library on the same flow cell.
- In cases where channels are mostly in the unavailable state and there is limited library, the flow cell can be washed to remove the blocks and reloaded with the same library.
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Recommendations
We recommend a library can be transferred a maximum of 2-3 times as with each recovery, a small amount of library is lost which may impact sequencing output and pore occupancy.
Highly viscous libraries, typically generated using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) are difficult to recover from the flow cell, leading to variable results. We recommend only transferring between PromethION flow cells, if required.
For the "Transfer a library between flow cells" method, we recommend transferring your library between flow cells immediately. Long term storage is not recommended following this method. The library can remain on the original flow cell in the device or stored in an Eppendorf DNA LoBind tube on ice for up to two hours, if required.
Longer term storage of a cleaned up library is only recommended when following the "Clean up and transfer a library between flow cells" method. The library can be stored at 4°C for short term storage or repeated use, for example, re-loading flow cells between washes.
We recommend avoiding transferring short fragment libraries (<2 kb) where possible as they are less efficiently transferred between flow cells which may negatively affect sequencing results.