Exit code 255 in Mageck Count

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1

Hi all,
i am having issues with the Mageck count feature in Galaxy.
I uploaded my CRISPR Screen NGS paired end data in fastsanger format, performed Trim Galore! and Mageck count.

The following error occurs:
Details
Execution resulted in the following messages:

Fatal error: Exit code 255 ()
Tool generated the following standard error:

INFO @ Tue, 14 Feb 2023 11:23:06: Parameters: /usr/local/tools/_conda/envs/mulled-v1-63b3fd1ab99fb7f24a87c2324cbe1d731c469003738e93222bc951289c466ee0/bin/mageck count -l /data/dnb07/galaxy_db/files/b/6/0/dataset_b60f594a-3cb6-4c70-887d-b879a2ec98c1.dat --fastq Trim_Galore__on_data_2__trimmed_reads_pair_1.fq Trim_Galore__on_data_2__trimmed_reads_pair_2.fq --sample-label Trim_Galore__on_data_2__trimmed_reads_pair_1,Trim_Galore__on_data_2__trimmed_reads_pair_2 -n output --pdf-report --keep-tmp --norm-method median
INFO @ Tue, 14 Feb 2023 11:23:06: Welcome to MAGeCK v0.5.8. Command: count
INFO @ Tue, 14 Feb 2023 11:23:06: Loading 312 predefined sgRNAs.
WARNING @ Tue, 14 Feb 2023 11:23:06: There are 0 sgRNAs with duplicated sequences.
INFO @ Tue, 14 Feb 2023 11:23:06: Parsing FASTQ file Trim_Galore__on_data_2__trimmed_reads_pair_1.fq…
INFO @ Tue, 14 Feb 2023 11:23:06: Determining the trim-5 length of FASTQ file Trim_Galore__on_data_2__trimmed_reads_pair_1.fq…
INFO @ Tue, 14 Feb 2023 11:23:06: Possible gRNA lengths:20
INFO @ Tue, 14 Feb 2023 11:23:06: Processing 0M reads …
INFO @ Tue, 14 Feb 2023 11:23:18: Read length:148,149,147,146,144,145,21,31,74,134,95,138,65,98,101,76,87,81,60,128,94,44,34,35,37,84,89,139,78,66,80,125,67,75,50,88,127,143,77,93,123,142,83,96,105,137,108,141,82,100,55,38,73,102,29,109,79,41,133,51,85,26,24,72,99,103,106,115,140,33,54,30,130,135,136,120,122,49,91,129,86,20,25,39,27,97,71,57,52,45,28,104,61,126,22,132,56,48,36,47,62,40,32,131,121,42,92,114,43
INFO @ Tue, 14 Feb 2023 11:23:18: Total tested reads: 100001, mapped: 0(0.0)
ERROR @ Tue, 14 Feb 2023 11:23:18: Cannot automatically determine the --trim-5 length. Only 0.0 % of the reads can be identified.

Link to history:
url: https://usegalaxy.eu/u/boegerm/h/input-only

I appreciate your help! Thank you very much!

2

Hello,

This is probably the most informative part of the error message. It looks like a content or parameter problem that you have now solved.

INFO @ Tue, 14 Feb 2023 11:23:18: Total tested reads: 100001, mapped: 0(0.0)
ERROR @ Tue, 14 Feb 2023 11:23:18: Cannot automatically determine the --trim-5 length. Only 0.0 % of the reads can be identified.

Galaxy tutorial with example data and usage: CRISPR screen analysis

Author reference: https://hpc.nih.gov/apps/MAGeCK.html

3

Hello Jennaj!

Thank you very much for your reply!

I do not fully understand how to solve the problem yet. Could you explain which parameters might have to be changed in order to solve the issue?

The error still occurs:
INFO @ Wed, 15 Feb 2023 12:06:29: Total tested reads: 100001, mapped: 6(5.999940000599994e-05)
ERROR @ Wed, 15 Feb 2023 12:06:29: Cannot automatically determine the --trim-5 length. Only 0.0059999400005999945 % of the reads can be identified.

Again thanks a lot, I appreciate your help! Best,

4

There were successful runs in the shared history, which means that the technical issues around trimming were resolved and now the issue is about content/parameters. The resources linked can help.

Also, you might want to toggle the tool to report a log (found under the Output Options) when exploring the data and different parameters to get more information about the processing details.

5

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