Summary table

Flow cell loading amount for ligation-based libraries

To maximise sequencing output, it is important that the pore activity is high: pores need to be kept filled with DNA, minimizing the time that they are “idle” in between strands. This means maintaining a high pore occupancy, ensuring that as many pores as possible are engaged with DNA at any given time. The benefit is twofold:

1. more data is produced per pore per hour.
2. the pore retention across the run is higher, resulting in more output from a flow cell.

We have found that achieving high pore occupancy with R10.4.1 (MinION, PromethION and Flongle flow cells) requires ~35-50 fmol of “good quality library”.

A “good quality library” can be atributted to the extent of the ligation of sequencing adapters during the library preparation. The presence of sequencing adapters at each end of the DNA molecules is the optimal conformation for tethering to the membrane and subsequent capture by the pore. Molecules without a sequencing adapter cannot be sequenced.

When loading a flow cell, the less library you load, the fewer “pore-threadable ends” are present on the flow cell. Pores will be “searching” for molecules for longer, and if the pores are not continuously sequencing then output could be compromised. It is important to note that we don’t observe a linear relationship between input onto the flow cell and sequencing yield, but underloading a flow cell can lead to reduced output.

For libraries of 1 kb and below we recommend an increased library load of 100 fmol of “good quality library” (see Figure 1), requiring starting with 200 fmol of input material. More DNA is required on the flow cell as short fragments are have a faster turn over sequencing time than longer ones.

For libraries 1-10 kb in length, in order to ensure you have sufficient library to load (i.e. 35-50 fmol) (see Figure 1), we recommended starting with 100-200 fmol of input material.

Figure 1. The relationship between amount of library loaded onto an Oxford Nanopore flow cell and the resulting pore occupancy that was obtained. a) For very short libraries (<1 kb) we find that pore occupancy is maximised when 100 fmol of library is loaded. b) We find that pore occupancy is maximised when >35 fmol of “good quality library” is loaded. Libraries were prepared using the Ligation Sequencing Kit V14 (SQK-LSK114) and loaded onto R10.4.1 flow cells.

For high-molecular weight (HMW), long fragment (>10 kb) libraries we recommend using mass rather than molarity for sample input and flow cell loading. We find that molar quantification of such samples is more difficult and more unreliable. For long fragment (>10 kb) libraries it is recommended to use a starting input of 1 µg. This is almost always sufficient to ensure optimal pore occupancy.

Figure 2. The relationship between HMW DNA input into the library preparation and pore occupancy. Sequencing libraries were prepared using the Ligation Sequencing Kit with various starting inputs of a gDNA sample comprising of long molecules, all of the yielded libraries were loaded onto PromethION flow cells. a) An input of ~0.5-1 µg was sufficient to obtain optimal pore occupancy. b) The fragment length distribution of the library.

Input amount

If you start with less than 1 µg HMW gDNA, then it is possible that output will decrease as pore occupancy deteriorates. However, even when starting with as little as 100 ng of high molecular weight DNA, we have observed outputs of >50 Gb from PromethION flow cells.

As you decrease input below 100 ng, pore occupancy significantly deteriorates and we would recommend that users consider shearing the DNA, or amplification (by PCR or MDA) to generate more template.

Shearing high molecular weight templates (for example using a Covaris g-TUBE or Megaruptor®) increases the number of molecules/ends to thread into the nanopores resulting in an increase pore occupancy and flow cell output. However, fragmenting the DNA to boost the output means that you are likely to observe a reduction in the observed read length.

We investigated the performance that can be obtained from human gDNA when starting with 100 ng, with and without shearing: it was observed that with shearing (e.g. using a Covaris g-TUBE), pore occupancy and output could be increased.

Figure 3. Output on PromethION flow cells with 100 ng of HMW human gDNA. Human gDNA was extracted from whole blood using the QIAGEN Gentra Puregene Blood Kit. The HMW gDNA was sheared using a Covaris g-TUBE. Libraries were prepared for sequencing with the Ligation Sequencing Kit using 100 ng of sheared and unsheared template DNA. The libraries were run on PromethION flow cells. Panel A: shearing the input gDNA decreases the read lengths that are observed in the subsequent sequencing. Panel B: the flow cell output (Gb) obtained from 100 ng of input is increased by shearing. Panel C: the increased shearing of the sample increases the pore occupancy (leading to the higher outputs from the sheared samples).

Change log

Version	Change
v1, Aug 2024	Initial publication