This protocol is a guideline to use alongside our automation protocols for PromethION to illustrate how to load, prime and wash up to 48 flow cells on a single device. Processing multiple flow cells can be time consuming and our protocols for sequencing kits are written for individual flow cells.

We estimate ~1 hour is saved using this protocol as it minimises pipette changes, including tip and volume alterations between steps and makes the most of the incubation time by completing each step per flow cell.

The user can adapt this protocol to any number of flow cells to fit their needs.

Priming and loading 48 flow cells takes approximately 1.5-2 hours. Washing, priming and re-loading 48 flow cells takes approximately 2-2.5 hours.

For information regarding the wash kit and the nuclease activity of the Flow Cell Wash Kit, please refer to the Flow Cell Wash Kit protocol.

We recommend using this as a guideline for how to prepare multiple flow cells alongside the relevant protocol for your sequencing kit.

To wash your flow cells, the Flow Cell Wash Kit XL (EXP-WSH004-XL) is required.

To reload the flow cell with a DNA library, the following additional reagents are required.

• Flow Cell Tether (FCT)
• Flow Cell Flush (FCF)
• Library Beads (LIB) or Library Solution (LIS)
• Sequencing Buffer (SB)
• Elution Buffer (EB)

These can all be found in the Sequencing Auxiliary Vials V14 (EXP-AUX003). If only flow cell priming reagents are required, these can be found in the Flow Cell Priming Kit V14 (EXP-FLP004).

Name	Acronym	Fill volume per vial (µl)	Cap colour	No. of vials	No. of uses
Wash Mix	WMX	150	Brown	1	48
Wash Diluent	DIL	20,000	White cap, brown stripe	1	48
Storage Buffer	S	25,000	White cap, yellow stripe	1	48

• Wash Mix (WMX) contains DNase I.
• Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
• The Storage Buffer allows flow cells to be stored for extended periods of time.

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

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

To load libraries into multiple PromethION Flow Cells as efficiently as possible, we recommend completing each step of the protocol per flow cell, following the same order of flow cells for each step.

Note: PromethION Flow Cells are shipped with light shields already installed. We recommend keeping the light shields on the flow cell to save having to intall them after loading your library.

For example, if flow cells in positions 1A, 1B and 1C are loaded into the device sequentially, the next step is completed in the same order.

Below is an animation for how we recommend loading a full column of eight flow cells. This can be applied to any number of flow cells. In the animation, we illustrate how to complete the step for one flow cell and highlight the next flow cell the step is performed on, to give a quick overview of the entire process.

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

An accumulation of pores in the 'recovering' state can occur ('unavailable' in the figure below). This reduces the rate of data acquisition due to fewer pores being available to accept and sequence strands. These pores can be reverted back to the 'active pore' state by pausing sequencing and washing the flow cell with the Flow Cell Wash Kit XL (EXP-WSH004-XL) (Figure 1). The wash step is only recommended when channels are lost to the 'recovering/unavailable' state. Further information is available in the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) protocol.

Figure 1. A flow cell has been loaded with a DNA library that has accumulated pores in the 'unavailable' state. The red asterisks indicate when sequencing has been paused and a wash step has been performed. After a wash step was completed, a significant number of pores reverted to the 'single pore' state and were available for sequencing.

• This protocol assumes that the flow cells have already had a DNA/RNA library run on them
• The aim is to remove most of the initial library and prepare the flow cells for the loading of a subsequent library
• The Wash Kit contains all solutions required for the removal of the initial library
• Data acquisition in MinKNOW should be stopped (if loading a new library or storing the flow cells), or paused (if loading more of the same library after the wash) during the wash procedure and also during subsequent library addition
• After the flow cells have been washed, a new library can be loaded, or the flow cells can be prepared for storage

Additional buffers are required for reloading a library following the washing of a flow cell. These can be found in the one of the following expansion kits:

• Sequencing Auxiliary Vials V14 (EXP-AUX003). This expansion contains vials of Sequencing Buffer (SB), Elution Buffer (EB), Library Solution (LIS), and Library Beads (LIB) with Kit 14 flow cell priming reagents: Flow Cell Flush (FCF) and Flow Cell Tether (FCT). This expansion is only compatible with our Kit 14 chemistry e.g. SQK-LSK114.

• Flow Cell Priming Kit (EXP-FLP004). This expansion contains both Kit 14 flow cell priming reagents required: Flow Cell Flush (FCF) and Flow Cell Tether (FCT). This expansion is only compatible with Kit 14 chemistry.

For our previous chemistries:

• Sequencing Auxiliary Vials expansion (EXP-AUX001). This expansion contains vials of Elution Buffer (EB), Sequencing Buffer (SQB), and Loading Beads (LB), additional to those found in DNA sequencing kits for our Kit 9 chemistry.

• Sequencing Auxiliary Vials expansion (EXP-AUX002). This expansion contains vials of Sequencing Buffer II (SBII), Elution Buffer (EB), Loading Solution (LS), and Loading Beads II (LBII), additional to those found in Kit 10 and 11 chemistry, such as:

  • Kit 10 e.g. SQK-LSK110
  • Kit 11 e.g. SQK-PCS111

• Flow Cell Priming Kit (EXP-FLO002). This expansion contains both flow cell priming reagents required: Flush Buffer (FB) and Flush Tether (FLT). This expansion is compatible with Kit 9, 10 and 11 chemistry.