Do you fragment your DNA? The efficiency may be better on a shorter DNA strands (as in 20 -50kb). Also, get rid of very short fragments using appropriate kits. Lower DNA amount may help as well. What you could also do, is get a set of flongles and try different approaches - will be much less expensive than ruining full size cells.

Aidas Nasevicius Appreciate your suggestion. We experimented with both fragmentation and size selection methods (eg. with circulomics), but observed limited improvement. The idea of using Flongles for further experimentation appears promising; thank you for the advice!

I recently did a couple of minion runs. I used the Circulomics kit for size exclusion of fragments less than 25kb. I had a good N50 ranging from 12kb to 22kb with 16-20GB of data in one run. The only thing I suggest is not to freeze and thaw your DNA. If possible, perform the DNA extraction and library preparation on the same day; if not, don't freeze the DNA (you may use 4 degree storage but don't go higher). I think it's better to have more DNA than recommended during library preparation. For higher data, make sure your flow cells are within warranty, with a standard number of pores available for sequencing after the loading library.

Anil Baniya Those are great results! May I ask you about the species from which you achieved these outcomes? We are encountering challenges specifically with amphibian DNA, despite our team's success in sequencing various organisms such as plants, mammals, and insects in the past.

Virag Nyiri I used nematode.