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New kit extends yields of flow cells

Wed 18th September 2019

Today we've released an updated Flow Cell Wash Kit which will be included in all starter packs and is also available to purchase separately on the Nanopore Store. The kit enables increased yields from existing flow cells.

The Flow Cell Wash Kit provides a highly effective means of removing a library that has been loaded onto a MinION or PromethION flow cell. Once washed, the flow cell can be re-used immediately, or stored for later usage.

Many users have been benefiting from adding a nuclease flush to their workflows, seeing increased throughput from flow cells as a result — the new Flow Cell Wash Kit provides a simple alternative to including a nuclease flush protocol in their workflow.

Minimal sample cross over

The Flow Cell Wash Kit is based on the use of a nuclease to digest and remove nucleic acid that has been loaded onto the flow cell. The washing procedure is very effective with as little as 0.1% of any previously loaded sample contaminating a subsequent run. Barcoding samples will allow the user to filter out any reads from the previous library that is still remaining on the flow cell after the wash.

Figure 1. The Flow Cell Wash Kit removes up to 99.9% of template molecules from the previous library. Where barcoded libraries are sequentially run without washing in between flow cell loads, significant contamination is observed from the previously loaded sample (a). However, washing the flow cell before loading the next sample shows that most of the previous sample has been removed (b). Barcoding samples that are to be sequentially run can help avoid cross-contamination.

No impact on read length

After nuclease treatment, and before storing or re-loading the flow cell, the nuclease is inactivated to prevent residual activity impacting future flow cell runs. We do not routinely observe any deterioration in read lengths in sequencing runs performed after the washing procedure (see figure 2).

Figure 2. The nuclease is efficiently inactivated before re-using or storing the flow cell. A sequencing library was prepared, and part of the library was loaded on to a flow cell (load #1). After a period of time, the run was stopped and a wash performed. The flow cell was re-loaded with more of the same library and the experiment re-started (load #2). The washing and re-loading steps were repeated (load #3). No difference in read length distributions was observed for the 3 library loads, suggesting very little residual nuclease activity is present after inactivation.

Improved flow cell output

In experiments where throughput is limited by the increase in pores in the recovering”/“unavailable” state, we have shown that output can be at least doubled by performing several wash steps over the lifetime of a flow cell (see figure 3 and 4).

 

Figure 3. A MinION flow cell has been loaded with a sequencing library that has resulted in an accumulation of channels in the “unavailable” state, leading to a decrease in the rate of data acquisition: after 18 hours, the flow cell has <200 single pores available for sequencing from a starting point of ~1600. A washing procedure was then performed after and the number of single pores increased to ~1000, with a significant number of the channels that had been lost to the “unavailable” state reverting to the “single pore” state.

Figure 4. Throughput observed from sequencing libraries 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 due to the accumulation of “unavailable” pores. In each case, throughput is more than doubled from the point of the first wash.

Find out more about the Flow Cell Wash Kit in the Community and the Nanopore Store.

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