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 cells

You 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 cells

You 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 cell

You 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

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.

• 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.

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION flow cells. Oxford Nanopore Technologies will replace any flow cell with fewer than 800 pores 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 in the Flow Cell Check document.

Ensure the compatible flow cell priming reagents are used for your libraries. These are required for all methods and are available with our sequencing kits. If extra reagents are required, they can be found in the following expansion kits.

For libraries prepared using Kit 14 chemistry, please ensure you are using the following reagents:

• Flow Cell Tether (FCT)
• Flow Cell Flush (FCF)
• For MinION flow cells only, we recommend using Bovine Serum Albumin (BSA)

Flow Cell Priming Kit (EXP-FLP004) contents:

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Flow Cell Flush	FCF	6	Clear cap, light blue lable	8,000
Flow Cell Tether	FCT	1	Purple	200

For libraries prepared using Kit 9, 10 or 11 chemistry, please ensure you are using the following reagents:

• Flush Buffer (FB)
• Flush Tether (FLT)

Flow Cell Priming Kit (EXP-FLP002) contents:

Name	Acronym	Cap colour	No. of vial	Fill volume per vial (μl)
Flush Buffer	FB	Blue	6	1,170
Flush Tether	FLT	Purple	1	200

Short Fragment Buffer (SFB) and Elution Buffer (EB) are both required for the "Clean up and transfer a library between flow cells" method. Both reagents are found in our sequencing kits but extra reagents can be found in the below kits.

Short Fragment Buffer (SFB) is available in the SFB Expansion (EXP-SFB001).

SFB Expansion (EXP-SFB001) contents:

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Short Fragment Buffer	SFB	Grey	4	1,800

Elution Buffer (EB) is available in the Sequencing Auxiliary Kit.

For libraries prepared using Kit 14 chemistry, please ensure you are using the following kit which also includes the flow cell priming reagents:

Sequencing Auxiliary Vials V14 (EXP-AUX003) contents:

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Elution Buffer	EB	Black	2	500
Sequencing Buffer	SB	Red	2	700
Library Solution	LIS	White cap, pink label	2	600
Library Beads	LIB	Pink	2	600
Flow Cell Flush	FCF	Light blue label	2	8,000
Flow Cell Tether	FCT	Purple	2	200

For libraries prepared using the Kit 10 or 11 chemistry, please use the following kit:

Sequencing Auxiliary Vials (EXP-AUX002) contents:

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Sequencing Buffer	SBII	Red	4	500
Loading Solution	LS	Pink sticker on cap	2	360
Elution Buffer	EB	Black	2	200
Loading Beads	LBII	Pink	2	360

For libraries prepared using Kit 9 chemistry, please use the following kit:

Sequencing Auxiliary Vials (EXP-AUX001) contents:

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
Sequencing Buffer	SQB	Red	6	300
Elution Buffer	EB	Black	2	200
Loading Beads	LB	Pink	2	360

For the "Clean up and transfer a library between flow cells" method, AMPure XP Beads (AXP) are required. These are available in many of our kits where the DNA library undergoes a clean up step. However, if extra beads are required, we recommend purchasing more from Beckman Coulter, Inc. (A63880).

The Flow Cell Wash Kit is required for the "Recover a library to replace on a washed flow cell" method.

Flow Cell Wash Kit contents (EXP-WSH004):

Contents	Volume (µl)	No. of tubes	No. of uses
Wash Mix (WMX)	15	1	6
Wash Diluent (DIL)	1,300	2	6
Storage Buffer (S)	1,600	2	6

• This protocol assumes that the original flow cell has been loaded with a DNA library prepared in accordance with the suitable protocol.
• The aim is to prepare the second flow cell and recover the library from the original flow cell for immediate transfer.
• Data acquisition in MinKNOW should be stopped during the recovery and transfer procedure.
• After the second flow cell has been primed, the library can be recovered and immediately transferred to the second flow cell.

• The aim is to recover and clean up your library before priming the second flow cell for loading the recovered library at a later date.
• The cleaned up library can be stored at 4°C for short term storage or repeated use, for example, re-loading flow cells between washes.
• Data acquisition in MinKNOW should be stopped during the recovery and transfer procedure.

• This protocol assumes that the original flow cell has been loaded with a DNA library prepared in accordance with the suitable protocol.
• The aim is to recover the library and wash the flow cell to reload the recovered library.
• Data acquisition in MinKNOW can be paused during the procedure.

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Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.