Fastqc errors with non-zero exit status 2

Question

I tried to run fastqc via a Snakemake command on my local computer. It didn’t work.

rule fastqc_raw:
     input:
         "raw/A.fastq"
     output:
         "output/fastqc_raw/A.html"
     shell:
         "fastqc {input} -o {output} -t 4"

It displayed this error:

Error in rule fastqc_raw:
jobid: 1
output: output/fastqc_raw/A.html   RuleException: CalledProcessError in line 13 of
/Users/01/Desktop/Snakemake/Snakefile: Command ' set -euo pipefail; 
fastqc raw/A.fastq -o output/fastqc_raw/A.html -t 4 ' returned
non-zero exit status 2.   File
"/Users/01/Desktop/Snakemake/Snakefile", line 13, in __rule_fastqc_raw
File "/Users/01/miniconda3/lib/python3.6/concurrent/futures/thread.py",line 56, in run

However the snakemake program did created DAG file that looks normal and when I used “snakemake --np” command, it didn’t display any errors.

Related to this- https://stackoverflow.com/questions/44616073/thread-py-error-snakemake — Manavalan Gajapathy, Oct 03 '18 at 19:24

score 1 · Answer 1 · edited Nov 27 '18 at 01:02

1

It looks like Snakemake did its job. It ran the command:

fastqc raw/A.fastq -o output/fastqc_raw/A.html -t 4

But the command returned an error:

Command ' set -euo pipefail; 
fastqc raw/A.fastq -o output/fastqc_raw/A.html -t 4 ' returned
non-zero exit status 2.

The next step in debugging is to run the fastqc command manually to see if it gives an error.

edited Nov 27 '18 at 01:02

beroe

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answered Oct 18 '18 at 15:31

Marmaduke

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score 1 · Answer 2 · answered Aug 07 '19 at 03:22

I hope you have gotten an answer by now but I had the exact same issue so I will offer my solution.

The error is in the

shell: "fastqc {input} -o {output} -t 4"

FastQC flag -o expects the output directory and you have given it an output file. Your code should be:

shell: "fastqc {input} -o output/fastqc_raw/ -t 4"

Your error relates to the fact that the output files have been output in a different location (most likely the input directory) and the rule all: has failed as a result.

Additionally, FastQC will give an error if the directories are not already created, so you will need to do that first.

It is strange as I have seen Snakemake scripts that have no -o flag in the fastqc shell and it worked fine, but I haven't been so lucky.

score 0 · Answer 3 · answered Jul 05 '21 at 22:54

An additional note: I can see you're using 4 threads there with the '-t 4' argument. You should specify this so Snakemake gives it 4 threads, otherwise I believe it will run with 1 thread and may fail due to lack of memory. This can be done like so:

rule fastqc_raw:
input:
    "raw/A.fastq"
output:
    "output/fastqc_raw/A.html"
threads: 4
shell:
    "fastqc {input} -o {output} -t 4"

Fastqc errors with non-zero exit status 2

3 Answers3