Readfish on prom

[hasindu2008]

I tried the https://github.com/LooseLab/readfish/commit/23dd37117bce576b99caf097e7711dc87d30fa0a for readfish on the PromethION. The installation went smoothly, however, when I tried on an actual run (not a play back, an actual run with a previously used flowcell), the flowcell position went to an 'unknown' state.

What I did:

pip install git+https://github.com/LooseLab/readfish@23dd37117bce576b99caf097e7711dc87d30fa0a
pip install git+https://github.com/nanoporetech/read_until_api@v3.0.0
pip install ont-pyguppy-client-lib==6.1.5

TOML file:

[caller_settings]
config_name = "dna_r9.4.1_450bps_fast_prom"
host = "ipc:///tmp/.guppy"
port = 5555
barcode_kits = ["EXP-NBD104"]
align_ref = "/data/readfish/hg38noAlt.idx"

[conditions]
reference = "/data/readfish/hg38noAlt.idx"

[conditions.0]
name = "ReadFish_v6_gene_targets.collapsed.hg38"
control = false
min_chunks = 0
max_chunks = 16
targets = "ReadFish_v6_gene_targets.collapsed.hg38.txt"
single_on = "stop_receiving"
multi_on = "stop_receiving"
single_off = "unblock"
multi_off = "unblock"
no_seq = "proceed"
no_map = "proceed"

Command:

readfish targets --device 5F --experiment-name "ReadFish" --toml ReadFish.toml --cache-size 3000 --batch-size 3000 --channels 1 3000 --port 9502

The system log is here: https://www.dropbox.com/s/wuuhr5i2cn2opxz/syslog.txt?dl=0

[alexomics]

I've only managed to get a flow cell into that state by starting readfish before the run has properly started. I typically begin readfish as the mux scan is ending. I'm out of the lab today, but will attempt to recreate tomorrow.

Do you have the control server or bream logs for the position that was affected, these should be under /var/log/minknow/5F/?

[hasindu2008]

Oh right. I launched it during the mux scan, my bad. I will reattempt it after the mux scan is completed - on Tuesday next week - as our PromethION is currently busy sequencing some other normal sequencing runs.

The 5F logs are here: https://www.dropbox.com/sh/cvogti3vyg1kk2z/AADIxy9_MxICCbKjnCDzSWl-a?dl=0

[hasindu2008]

@alexomics This time I launched readfish after mux scan and it successfully communicated with read fish and printed the usual output.

Is it possible to disable the barcode-aware readfish and run normal readfish for the promethion? What change should I do to my toml?

[alexomics]

That should be just removing the barcode_kits from the TOML and running readfish targets …. The align_ref will still need to be set on the caller_settings table.

[hasindu2008]

Thanks for the quick response. Will soon give a try and let you know.

[hasindu2008]

Seems to be running. This is a check using a bad flowcell so the pore count is less. Any idea what this red colour state in the end reason plot is?

[mattloose]

We are told the red is Minknow misclassifying those reads. We've seen it too and it isn't anything to be concerned about.

[hasindu2008]

Ok Thanks. What is the recommended amount of DNA to be loaded for R9.4.1 LSK110? Like for MinION flowcells is it recommended to be washed every 24 hours and reloaded?

[mattloose]

Definitely flush and reload every 24 hours. We tend to overload a little for lsk110 r9.4 - so somewhere in the range of 1.2 - 1.5 x the normal number of molecules.

You are trying to maximise occupancy throughout the run.