Hi all, I am looking at using Nanopore sequencing using the MinION to sequence a library of small (5kb), barcoded plasmids (~5,000 plasmids in total). I am generating ~100 plasmids at a time, and rather than waiting until all 5,000 are ready I would like to sequence them as they are generated. The ligation sequencing kit we would like to use is stated to generate 6 libraries, however given that I do not require deep coverage of my plasmids and that the plasmids are relatively small, I don't want to waste an entire library kit on a small (< 6 hr) run. Does anyone have any experience using half the recommended DNA concentration (or less) to generate smaller libraries to run on the minION? Thanks!
Hi Kshitij, if I understand correctly you would like to generate smaller libraries to save the budget for the ligation kit. I have not done this before, but from your description, if you add a half-recommended kit, your DNA concentration per library is actually doubled. You may need to add the same DNA concentration as recommended but with a half volume. Then, theoretically speaking, the adaptor ligation efficiency should be the same.
Another thing that needs to consider is flow cell durability and reliability after washing, the flow cell recovery percentage after a first-time wash is ~70% according to Nanopore. However, there are people who said it would be as lower as 25% (see, https://www.researchgate.net/post/Are_nanopore_flow_cells_reusable).
Last but not least, DNA is quite stable, if it is a budget concern, the best solution is sequencing 5000 pre-barcoded plasmids, which save you tons of time of preparing the library and washing the flow cell, and money! However, I do not know if it is practical to prepare all of your samples in one day. If you can manage to pool all samples together, I think you can get good coverage (750 Mbp/25Mbp = 30X) by just running once for 72 hours.
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