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novoalab / Nano-tRNAseq

Quantitative analysis of native tRNA populations using direct RNA nanopore sequencing (Lucas*, Pryszcz* et al., Nat Biotech 2023)
GNU General Public License v3.0
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Config settings for Promethion? #7

Closed lkwhite closed 9 months ago

lkwhite commented 1 year ago

I'm trying to make changes to config file to permit short mode sequencing and/or simulation on a Promethion. A first naive attempt was to just try adding Promethion flow cells to flow_cells.toml and then add "FLO-PRO002_short" to sequencing_MIN106_RNA_short.toml. However, when we actually try to start a run with this short read mode, we get through the flow cell check and then almost immediately drop to zero pores. My suspicion is that one or more of the settings in this file (target temperature? min/max voltage?) are MinION specific and incompatible with Promethion settings, causing the pores to register as nonfunctional once the run starts.

Have you all played with settings for the Promethion at all? I shot an email to ONT customer support too, but that's always hit or miss.

lpryszcz commented 1 year ago

Hi Laura, custom flowcell configurations didn't work with actual sequencing for us. You need to run sequencing with default config, save bulk file and run sequencing simulations with alternative config as described here. I know, it's troublesome, but we were unable to make it easier... We haven't tried it with PromethION yet. Please let us know if it worked for you. Greetings from Barcelona!

lkwhite commented 1 year ago

Oh, interesting! I saw the note that custom configurations can be used only with sequencing simulations, but naively tried a run in short mode using a Flongle flow cell and it worked just fine for sequencing ~& base-calling~ in real time. I wonder why?

Edit: looks like we didn't basecall in real time, just generated fast5s, but we got 32k reads from an expired flongle with an N50 of 143, so seemingly successful capture of short things.

lpryszcz commented 1 year ago

hah, that's peculiar. we neither tried with flongle I guess. or if we did, we just saved bulk and reprocess it. good to know!