Questions

[mkdir]

Hello,

I am a newbie to Oxford Nanopore sequencing technique. I have a couple of questions. How many active nanopores will be good enough? Is the account activation of metrichor required to run MinION seq? Thank you very much.

[Markiyan]

Nanopore R9 2016/05, Some real lifetime experience.

Hello,
I am a newbie to Oxford Nanopore sequencing technique. I have a couple of questions. How many active nanopores will be good enough?

Hmm..... that is a very good question....

I would say at least 50% of the active pores would be good (>1K active in all 4 multiplexes), but the main thing is, that this number tends to fall by 4-5 times after the first 24h of the run, and than follows the exponential decay curve (despite library and fuel mix top-ups) for the R9 flowcells made at the end of May. So I would treat the quoted 72h flowcell lifetime as very optimistic. Hopefully it would improve in the future.

Anyway, if you get 500M events from your first 24/48h run with a good library (20Kbp average fragment size) - than you had achieved your objective :-)

Is the account activation of metrichor required to run MinION seq? Thank you very much.

No, there is no requirement to run the cloud-based base calling during minion run, you can save the fast5 files, and basecall them later.
Also you can try the nanocall program for 1D data.:

https://github.com/mateidavid/nanocall

https://github.com/mateidavid/nanocall-analysis

[GenoMax]

No, there is no requirement to run the cloud-based base calling during minion run, you can save the fast5 files, and basecall them later.
Also you can try the nanocall program for 1D data.:

https://github.com/mateidavid/nanocall

https://github.com/mateidavid/nanocall-analysis

But do you recommend that for someone new (sounds like you have some experience with Nanopore)? Shouldn't they be using the standard tools that Oxford provides for their initial runs?

[Markiyan]

But do you recommend that for someone new (sounds like you have some experience with Nanopore)? Shouldn't they be using the standard tools that Oxford provides for their initial runs?

Yes, of course they should use the stock stuff first, but if they are new to the platform, it doesn't mean that they are new to the field of sequencing/bioinformatics.

IMHO: Usually it is good idea to know about other opensource alternative projects, especially if the user seems to be interested in the off-line data processing.

[mkdir]

Hi Markiyan, Thank you very much for explaining the details. These help a lot.

Hmm..... that is a very good question....

I would say at least 50% of the active pores would be good (>1K active in all 4 multiplexes), but the main thing is, that this number tends to fall by 4-5 times after the first 24h of the run, and than follows the exponential decay curve (despite library and fuel mix top-ups) for the R9 flowcells made at the end of May. So I would treat the quoted 72h flowcell lifetime as very optimistic. Hopefully it would improve in the future.

Anyway, if you get 500M events from your first 24/48h run with a good library (20Kbp average fragment size) - than you had achieved your objective :-)




No, there is no requirement to run the cloud-based base calling during minion run, you can save the fast5 files, and basecall them later.
Also you can try the nanocall program for 1D data.:

https://github.com/mateidavid/nanocall

https://github.com/mateidavid/nanocall-analysis

[mkdir]

Hi GenoMax, thank you very much for reminding.

But do you recommend that for someone new (sounds like you have some experience with Nanopore)? Shouldn't they be using the standard tools that Oxford provides for their initial runs?