The Flow Cell Wash Kit allows sequential runs of sequencing libraries on the same flow cell. It works by flushing out and digesting the DNA library currently on the flow cell, before providing fresh sequencing buffer and library.

Using the Flow Cell Wash Kit provides the opportunity to utilise the same flow cell a number of times, maximising the available run time on your flow cell. The number of flow cell wash and reuses will depend on various factors. These numbers typically range from 3-6 flow cell wash and reuses. These factors include:

• the amount of output generated per sequencing run: the total flow cell output is finite.
• the quality and concentration of the library loaded: best results are achieved by following the recommendations in our protocols on sample quality and loading amounts.
• time to answer requirements: as the number of pores on the flow cell decreases, the time taken to generate a fixed amount of data will increase. You can continue to run your flow cell with pore counts below the warranty number, so long as you are aware and happy with the reduced rate of data output.

Following the wash step, you have two options for reusing your flow cell:

• Load a subsequent or new library on your washed flow cell to begin sequencing straight away.
• Introduce Storage Buffer (S) from the Flow Cell Wash Kit into the flow cell, allowing you to store the washed flow cell at 2–8°C for later use.

Complete step-by-step instructions for using the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) can be found in our protocol in the documentation section of the Oxford Nanopore Community:

• Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) protocol

The Flow Cell Wash Kit contains a nuclease that digests the library DNA strands and sequencing adapters for effective removal from the flow cell. The nuclease wash has been demonstrated to be up to 99.9% effective in removing libraries from the flow cell.

Figure 1. 3 libraries containing 4 barcodes each were sequenced subsequently using EXP-WSH004. This was tested across 8 MinION and GridION R10.4.1 Flow Cells, and across 15 PromethION R10.4.1 Flow Cells by loading Lambda DNA (LMD from EXP-CTL001) libraries, prepared with the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96). Each flow cell load comprised an equal mixture of 4 barcodes per sequencing run. Sequencing runs were performed for 45 minutes. For MinION and GridION 14/16 runs had <99.9% barcode carryover between sequencing runs following a flow cell wash, with the outlier in run 2 being due to user error. For PromethION 29/30 runs had <99.9% barcode carryover between sequencing runs following a flow cell wash.

For optimal results, where possible, we recommend barcoding your samples using one of our barcoding kits. This will ensure if there is any carryover of trace libraries between sequencing runs, these are easily differentiated.

Successful demultiplexing of DNA reads has been demonstrated by Oxford Nanopore's internal development:

Figure 2. 8 MinION and GridION R10.4.1 Flow Cells were loaded and sequenced for 45 minute runs, using the Lambda DNA (LMD from EXP-CTL001) libraries, prepared with the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) as seen in figure 1. Between each run, a flow cell wash was performed using EXP-WSH004 and fresh sequencing libraries were loaded for the next sequencing run. Section A displays an overview of the experiments carried out. Section B displays the output of each sequencing run across all flow cells. Section C displays pore retention across each flow cell for the flow cell QC checks performed before the first sequencing run; after all sequencing runs were completed, the flow cell was washed and Storage Buffer (S) was introduced; and finally follwing overnight storage with Storage Buffer (S). Section D displays barcode carryover across each flow cell for sequencing runs #2 and #3, each after a flow cell wash.

We found that all 8 flow cells successfully accommodated 3 Flongle-style sequencing runs with comparable flow cell output. All flow cells retained sufficient pores to accommodate further sequencing runs. 5/8 flow cells retained more pores than the new MinION and GridION Flow Cell warranty (800 pores) over the course of the experiment, including after storing at 4ºC overnight. All flow cells retained more pores than the current Flongle flow cell warranty (50 pores). Sample carryover between sequencing runs was minimal (<0.1% except for one run, due to user error).

This demonstrates that users can achieve at least 3 Flongle-style sequencing runs on a MinION Flow Cell. For ease of use, we recommend to use the Run until function in MinKNOW to specify the amount of data per sequencing run to optimise flow cell usage across multiple uses.

For users who wish to use barcoding to run multiple libraries at one time rather than washing the flow cells, please see the barcoding kits we have available in the Oxford Nanopore Store.

During sequencing, an accumulation of pores in the Unavailable state (Figure 3) may be observed, causing the rate of data acquisition to decline as fewer pores are available to accept and sequence strands. We have demonstrated that in these circumstances, pores can be reverted to the Pore available state by pausing sequencing and washing the flow cell with the nuclease in the Flow Cell Wash Kit. In Figure 3, the asterisks indicate where sequencing has been paused and the flow cell washed.

Note: If the sequencing run is paused in MinKNOW for the flow cell wash, you will only see the restoration of sequencing pores after a new pore scan has been performed.

Figure 3. Pore states observed on a MinION and GridION Flow Cell before and after wash steps are performed. The flow cell has been loaded with a sequencing library that has resulted in an accumulation of pores in the Unavailable state, leading to a decrease in the rate of data acquisition. The red asterisks indicate when a wash step has been performed. A significant number of the pores that had been lost to the Unavailable state have reverted to the Pore available state and are available for sequencing once again.

The wash step is recommended where sequencing channels are lost to the Unavailable state (Figure 3). In circumstances where channels have been lost by other means, for example Saturated, the wash step is not effective at reverting channels to the Pore available state. If your sample is known to block and cause saturation of the flow cell, we recommend loading a fresh library on a new flow cell. If you do not have excess library to load on a new flow cell, you can recover the library and reload on a new flow cell following the method in the Library recovery from flow cells protocol.

In experiments where output is limited by the increase in pores in the Unavailable state, we have shown that output can be improved by performing several wash steps over the lifetime of a flow cell. Figure 4A shows the output obtained from a PromethION Flow Cell loaded with a library of DNA extracted from chicken - Gallus gallus, where unavailable pores increased over the course of the experiment, and so flow cell washes were performed to unblock the pores (Figure 4A). In each case, the use of multiple washes allowed for an improvement of the output from the flow cell, without any compromise in observed read length (Figure 4B).

Figure 4. Section A: Output observed from Gallus gallus sequencing library run on a PromethION Flow Cell. The arrows indicate the timing of each wash step: wash steps were performed at the point where the rate of data acquisition started to slow down. In each case, output is more than doubled from the point of the first wash. Section B: Read length of the Gallus gallus sequencing library was recorded before the first wash (load #1) and then again after the first and second washes (load #2 and #3, respectively). No decrease in read length is observed.

Note: The Flow Cell Wash Kit is compatible with Q-Line and R10.4.1 flow cells. The Flow Cell Wash Kit can also be used with our RNA flow cells to flush out RNA libraries but will not remove RNA-related blocking and restore nanopores.