This know-how document provides information for transitioning from a Flongle to a MinION Flow Cell. It compares the two flow cell types in terms of key differences, cost per flow cell versus cost per sample and genome sizes. It also describes ways of maximising the sequencing output from MinION Flow Cells and troubleshooting pore loss.

The MinION Flow Cell does everything the Flongle Flow Cell does, with improved performance across the board: higher output, larger sample capacity, longer warranty, can be washed and reused, greater pore redundancy, and a lower cost per sample. Both Flongle and MinION use the same library preparation kits, so you can transfer your methods directly across.

The table below compares the two flow cell types:

	Flongle	MinION
Channels	126	512
Pores per channel	1 (126 total)	4 (2,048 total)
Typical output*	~1.2 Gb	~24 Gb
Warranty	4 weeks	12 weeks
Flow cell pore warranty	Minimum 50 pores	Minimum 800 pores
Reuse	Single use	Washable, typically 3–6x
Devices	MinION / GridION	MinION / GridION

^*Outputs using PCR E.coli and starting with 1,500 pores on a MinION Flow Cell and 80 pores on a Flongle Flow Cell.

A MinION Flow Cell costs more upfront, however, it supports up to 96-plex barcoding, and can typically be washed and reused 3–6 times. This spreads the cost of a single flow cell across far more samples and far more data, lowering both the cost per sample and the cost per Gb compared with a single-use Flongle.

Flongle still has the lowest upfront cost per flow cell. However, for many workflows, once output, multiplexing, and flow cell wash and reload are accounted for, MinION proves to be more economical than Flongle.

Every sequencing and barcoding kit that you currently use with Flongle is also compatible with MinION Flow Cells. Therefore, library preparation uses the same kits and same barcodes.

You can run the same small libraries on a MinION, reaching the data target faster and keeping headroom for further libraries. For a 2 Gb target, 126 pores take ~12 hr on a Flongle (single use), while 512 pores take ~3 hr on a MinION. You can then stop the sequencing run early (MinKNOW Run until) and wash and reuse or store the flow cell for the next sample, so capacity is not wasted.

MinION Flow Cells run on the same MinION Mk1B, MinION Mk1D, and GridION devices that you already use for Flongle. Therefore, you do not need to purchase any new instrument or additional components.

In fact, you need one fewer component; the Flongle adapter is not required for MinION Flow Cells. The flow cell inserts directly into the device.

A simple way of comparing the two flow cell types is to determine how many samples of a given genome size can fit on each flow cell at 30x coverage.

This assumes perfect distribution of each genome on flow cells starting with 1,500 pores for a MinION Flow Cell and 80 pores for a Flongle Flow Cell.

The calculation:

1. Starting pores — output scales with the number of pores available at the start of the run.
   In the PCR E. coli example, a 1,500-pore MinION delivered ~24 Gb and an 80-pore Flongle delivered ~1.2 Gb.

2. Data needed per sample = genome size x target coverage
   e.g. 10 Mb genome x 30x coverage = 300 Mb = 0.3 Gb per sample

3. Samples per flow cell = total flow cell output ÷ data per sample
   e.g. MinION 24 Gb ÷ 0.3 Gb = 80 samples

In summary, the MinION Flow Cell carries approximately 18x more pores than a Flongle Flow Cell. This translates to proportionally more samples, or deeper coverage of the same samples on a MinION Flow Cell compared with a Flongle Flow Cell.

The table below compares the genomes per flow cell type for various organisms:

Organism	Genome size	Data per sample (30x coverage)	Genomes per Flongle Flow Cell (1.2 Gb)	Genomes per MinION Flow Cell (24 Gb)
Bacterium (E. coli)	~5 Mb	0.15 Gb	8	160
Yeast (S. cerevisiae)	~12 Mb	0.36 Gb	3	66
Fungus (N. crassa)	~40 Mb	1.2 Gb	1	20
Nematode (C. elegans)	~100 Mb	3.0 Gb	0	8
Insect (D. melanogaster)	~140 Mb	4.2 Gb	0	5
Plant (rice, O. sativa)	~390 Mb	11.7 Gb	0	2

Oxford Nanopore barcoding kits allow multiple libraries to be pooled and run together on a single flow cell. Each sample is tagged with a unique barcode, pooled for sequencing and separated again during analysis (demultiplexing).

As a result of the increased performance of the MinION Flow Cell, you can use the compatible barcoding kits, Rapid Barcoding Kit 24 or 96 V14 (SQK-RBK114.24/96), Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24/96), Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) or 16S Barcoding Kit 24 V14 (SQK-16S114.24) to support up to 96 samples on a single flow cell. When dividing the run cost across all samples, this dramatically lowers the cost per sample, while still providing sufficient data output per sample for most applications.

Washing increases the total usable output by unblocking pores. An accumulation of pores in the unavailable state due to blocking, causes the rate of data acquisition to decline. Removing the DNA blocking these channels can revert them to the pore available state, increasing the sequencing life of the flow cell.

You can wash and reuse MinION Flow Cells, whereas Flongle Flow Cells are for single use only.
Refer to the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) for instructions on washing the MinION Flow Cell.

You can use both barcoding and wash kits to further increase the number of samples that can be sequenced on one flow cell. The Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) allows sequential runs of different sequencing libraries on the same flow cell.

By barcoding each run and washing between runs, one flow cell can process several multiplexed batches in sequence. For smaller genomes, this means up to 96 samples per run, across typically 3–6 wash and reload cycles.

Note: The nuclease in the flow cell wash is up to 99.9% effective at removing libraries, but not 100%. If carryover between runs is likely to affect your downstream analysis, use a different set of barcodes after each wash, so that any residual library from the previous run can be identified and filtered out at demultiplexing.

You can use barcoding, washing and reloading to maximise samples and outputs for clinical applications, in laboratories with strict clinical requirements around zero carryover. We still recommend using the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) to wash and reuse the same flow cell several times, maximising available run time and improving output.

To manage carryover in clinical conditions, we recommend using a different set of barcodes for each run, after each wash. As the nuclease in the flow cell wash is only 99.9% effective in removing libraries, the use of fresh barcodes will allow any trace library from the previous run to be easily differentiated at demultiplexing and filtered out rather than being misassigned. This will enable efficient bioinformatic analysis while maintaining stringent contamination controls.

Pore loss is usually related to handling rather than the storage interval itself. If there is significant pore loss between the first 1–2 washes, this may be related to the flow cell.

Follow the recommendations below:

• Check that the flow cell has a reasonable number of available pores before washing. The Flow Cell Wash Kit will not be able to recover pores from a flow cell with minimal pores at the end of sequencing.

• Wash the flow cell promptly after the run ends. Do not leave the DNA library on the flow cell.

• Add storage buffer to the flow cell immediately after washing and store at 2–8°C.

• Complete all washes and reloads within the 12-week warranty window of the flow cell; sequencing beyond it may lead to greater pore loss and falls outside the warranty.

It is known that ligation sequencing chemistry provides cleaner sequencing with improved pore occupancy and yields compared with rapid chemistry. The reduced purification during rapid library preparation and the use of a transposase may lead to increased blocking of pores earlier in sequencing. This affects both flow cell types but is more pronounced on the Flongle. Where output and pore longevity are more important than the speed of library preparation, use the Ligation Sequencing Kit V14 (SQK-LSK114) or the Native Barcoding Kit 24 or 96 V14 (SQK-NBD114.24/96). Refer to the Chemistry technical document for more information.