Nanopore sequencing of labelled DNA

Question

[Jean-Karim Heriche]

We have DNA which has incorporated fluorescent nucleotide analogues. Questions are:
1- Do fluorophore-labelled nucleotides tend to clog the pores? If this happens, can the data still be used to some extent?
2- What base caller would be recommended?
3- Assuming there's no ready-to-go base caller for our data, are there tutorials/pointers on how to train a base caller?
4- If we are only interested in the lengths of labelled DNA fragments, is there a path to get this info that maybe doesn't involve base calling?

[GenoMax]

@Jean-Karim this may be best posted on ONTs' nanopore forum (when I last checked it needed an invite to join, in case you don't have an account). Please post the solution here if/when you get one.

[Jean-Karim Heriche]

OK. Will do. Thanks.

[WouterDeCoster]

I don't really have good answers for the other points, but for question 3 I would suggest having a look at sloika. Do you have an idea about the size of the fluorophores? I know BrdU sequencing is not a problem, but that's not quite the same.

[Jean-Karim Heriche]

Thanks for the pointer to sloika.
I don't know the exact size but the molecule is on the order of 1200-1300 Da.

Answer 1

[Jean-Karim Heriche]

Coming back to this for people following: there's no sign of pores being blocked but no indication that labelled DNA is going through the pores (there's no difference between labelled and unlabelled samples).
Anyway it looks like there may be a better way to get at what we need using optical replication mapping.