SquigglePlot command is not working

[TTT16]

Hi,
I tried the SquigglePlot command, and it gave me error like below. Could you please let me know how to fix it?

python /cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py -s /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit/SRR8767348_squiggle-pull.tsv.gz --raw_signal --save SRR8767348_squigglePlot.png --save_path /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit

Error:

Cannot convert the signal file. Will plot the values provided as is.
Traceback (most recent call last):
File "/cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py", line 474, in
main()
File "/cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py", line 198, in main
for l in sz:
File "/cm/local/apps/python3/lib/python3.9/codecs.py", line 322, in decode
(result, consumed) = self._buffer_decode(data, self.errors, final)
UnicodeDecodeError: 'utf-8' codec can't decode byte 0x8b in position 1: invalid start byte

[Psy-Fer]

Hello,

That looks like you are putting a gzipped signal file to SquigglePlot. It currently doesn't accept gzipped signal files. Try unzipping it and trying again.

James

[TTT16]

Hi James,
I tried to unzip the tsv file, and do it again, but it still gave me error. How can I fix it? THank you!

[tmhtxt53@cn001 ~]$ python /cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit/SRR8767348_squiggle-pull.tsv --raw_signal --save SRR8767348_squigglePlot.png --save_path /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit
error: unrecognized arguments: /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit/SRR8767348_squiggle-pull.tsv
usage: SquigglePlot.py [-h] [-p F5_PATH | -s SIGNAL | -w SLOW5 | -i IND [IND ...]] [-r READID] [--single] [--head] [--raw_signal] [-n NUM]
[--lim_hi LIM_HI] [--lim_low LIM_LOW] [--plot_colour PLOT_COLOUR] [--save SAVE] [--save_path SAVE_PATH] [--no_show] [--dpi DPI]

SquigglePlot - plotting the raw signal data after (optional) conversion to pA

[Psy-Fer]

Hello,

You are missing the argument in front of the signal file in your command.

It should be

python /cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py -s /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit/SRR8767348_squiggle-pull.tsv --raw_signal --save SRR8767348_squigglePlot.png --save_path /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit

James

[TTT16]

Hi, sorry for my mistake. I did again like you suggested, and it gave me warning/error like below:

[tmhtxt53@cn001 ~]$ python /cm/shared/apps/squigglekit/1.5.1a/SquigglePlot.py -s /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit/SRR8767348_squiggle-pull.tsv --raw_signal --save SRR8767348_squigglePlot.png --save_path /condo/kisslab/tmhtxt53/GridIon/m6Apaper_data/SquiggleKit
Cannot convert the signal file. Will plot the values provided as is.

And endless output png files as here. Is it supposed like this?
17993bb4-e99a-47c3-9f6e-7310e0c3500a_dpi_100_SRR8767348_squigglePlot.png
179c1b72-19c1-4c1b-86d6-b8c914d763fd_dpi_100_SRR8767348_squigglePlot.png
17ad5912-0c03-47fd-8328-469674fc909c_dpi_100_SRR8767348_squigglePlot.png
17b14433-fb0f-4a7a-ba02-32e74303220c_dpi_100_SRR8767348_squigglePlot.png
17c96a26-658b-4b64-9f1a-b0eb2ba1a7c3_dpi_100_SRR8767348_squigglePlot.png
17cf684d-773e-4614-a2cb-46ebf5c7a054_dpi_100_SRR8767348_squigglePlot.png
17d49fad-d1a6-48d3-97ec-49da71ae6a1e_dpi_100_SRR8767348_squigglePlot.png
17d865ba-8bda-4ca9-9033-4a254988a3b1_dpi_100_SRR8767348_squigglePlot.png
17e3654d-ed4e-4a17-9af0-240b28c7aac9_dpi_100_SRR8767348_squigglePlot.png
182cac11-e9e8-44a9-ac7a-dbf75d4f530a_dpi_100_SRR8767348_squigglePlot.png
184770d8-09e5-443e-ab51-f8c441bca726_dpi_100_SRR8767348_squigglePlot.png
185c44f4-c1c1-46bd-8253-f3e79cf85731_dpi_100_SRR8767348_squigglePlot.png
186606b3-c85a-43d5-8c6a-e0054860c7e9_dpi_100_SRR8767348_squigglePlot.png
187a06cf-511a-43b1-95e4-6aacc090463d_dpi_100_SRR8767348_squigglePlot.png
188e622d-564c-4345-8c6a-71b441c9f0a5_dpi_100_SRR8767348_squigglePlot.png
188e9b29-fbc4-4c7e-90fb-4cfa7090430c_dpi_100_SRR8767348_squigglePlot.png
18936c5f-dfe8-4bf7-af53-63a831fc319b_dpi_100_SRR8767348_squigglePlot.png
189ef11f-5df8-4d9f-b435-06a794d36ca3_dpi_100_SRR8767348_squigglePlot.png
18aa4ed0-b74f-4c2f-a96d-3078a1c58258_dpi_100_SRR8767348_squigglePlot.png
18ad70f9-4620-4e4e-b09a-1a8ce48367bb_dpi_100_SRR8767348_squigglePlot.png
18c2ee8f-7e6c-43fc-9865-aaf4a4f4a5c9_dpi_100_SRR8767348_squigglePlot.png
18c5831f-0f78-4994-8fd5-400adcac1336_dpi_100_SRR8767348_squigglePlot.png
18c9ff7c-bdfe-4914-bcb1-1ee8cf507f15_dpi_100_SRR8767348_squigglePlot.png
18cff98b-349e-46b3-8b85-5b687db47785_dpi_100_SRR8767348_squigglePlot.png
18f44dd4-30ce-46a0-839f-f9be0f9b4c6c_dpi_100_SRR8767348_squigglePlot.png
190d3dd1-0d36-45db-88bd-de7e24d32c02_dpi_100_SRR8767348_squigglePlot.png
193081e4-57cc-4a8d-a055-d74ee0dcb482_dpi_100_SRR8767348_squigglePlot.png
1934a71e-a832-4544-8744-f7e2adba12cc_dpi_100_SRR8767348_squigglePlot.png
193ee89f-f96e-4695-a725-59451e6a3391_dpi_100_SRR8767348_squigglePlot.png
19614e75-db83-4641-8e2a-086c77b11489_dpi_100_SRR8767348_squigglePlot.png

Thanks!

[TTT16]

And, one of png file like this:

How do I know where is A, U, C, G or modified nucleotides?
Thank you

[Psy-Fer]

Hello,

SquiggleKit does not do base-to-signal alignment or modification detection. It was built primarily to allow users to visualise the signal data to get more comfortable with the data to use in other methods, or to visually check strange results.

If you are looking for base-to-signal alignment, please use https://github.com/hiruna72/squigualiser by Hiruna from our group.
If you are looking for RNA mods, try this https://github.com/GoekeLab/m6anet which we helped get working for RNA004.

The output for RNA004 being in fast5 format from MinKNOW is a bug by ONT. I think they have fixed it now. Either way, nanopore has a conversion tool for fast5->pod5 in their pod5 tools here https://github.com/nanoporetech/pod5-file-format/

or if you want to use slow5, we have a fast5->slow5 in slow5tools https://github.com/hasindu2008/slow5tools
and if you need it, a pod5<->slow5 converter with https://github.com/Psy-Fer/blue-crab

I hope this helps.

James