Quality-, adapter- and RRBS-trimming with Trim Galore!

[LindsayR]

Thanks for helping Felix. A simple typo on my end unfortunately. Problem solved!

[create.share]

For those with weaker heart (those that cannot use complex scripts in Linux and need a graphic interface) here is another (free) program for trimming qualities:

An efficient SFF/FastQ viewer and editor (GUI)






The gray/green curves in the second graphic shows the average quality before and after trimming the low quality ends.


Only works on Fasta, FastQ, SFF for the moment.
Sorry.

[padmoo]

Hi everyone,

not sure if this is the right thread but I'll give it a go:

I'm trying to get rid of adapters in my sequences. There aren't many with adapters but I'd like to save what I can.

When I run my command I get the following error:

==================================================================
Your job looked like:

------------------------------------------------------------
# LSBATCH: User input
/gpfs/scratch/cbh12wsu/trim/trim_galore -phred33 --illumina --paired -phred33 --path_to_cutadapt /gpfs/scratch/cbh12wsu/trim/cutadapt.py -o adaptrim.fastq -length 50 1_L.fastq 2_R.fastq
------------------------------------------------------------

Exited with exit code 2.

The output (if any) follows:

Path to Cutadapt set as: '/gpfs/scratch/cbh12wsu/trim/cutadapt.py' (user defined)
File "/gpfs/scratch/cbh12wsu/trim/cutadapt.py", line 99
print(rest, match.read.name, file=self.file)
^
SyntaxError: invalid syntax
Cutadapt seems to be working fine (tested command '/gpfs/scratch/cbh12wsu/trim/cutadapt.py --version')
File "/gpfs/scratch/cbh12wsu/trim/cutadapt.py", line 99
print(rest, match.read.name, file=self.file)
^
SyntaxError: invalid syntax
Failed to write to file '1_L.fastq_trimming_report.txt': No such file or directory
==================================================================

Does indicate a problem with the cutadapt.py?

Thanks!

[fkrueger]

Hmm, when you just type "/gpfs/scratch/cbh12wsu/trim/cutadapt.py" on the command line do you see something like:

cutadapt version 1.8.1
Copyright (C) 2010-2015 Marcel Martin <[email protected]>

cutadapt removes adapter sequences from high-throughput sequencing reads.

Usage:
    cutadapt -a ADAPTER [options] [-o output.fastq] input.fastq

?

[padmoo]

Hi Felix,

thanks for replying so quick!

I get the following error when I try your command:
File "/gpfs/scratch/cbh12wsu/trim/cutadapt.py", line 99
print(rest, match.read.name, file=self.file)
^
SyntaxError: invalid syntax

I think I should mention that I made cutadapt.py an executable. And copied it from it's original folder into the trim folder.

[fkrueger]

Cutadapt probably needs its internal structure to be intact, can't you just do a normal installation and then point to the cutadapt executable (without renaming it to .py?)

[padmoo]

I put it back where it belongs and tried again. Same error as before.

With normal installation you mean the pip command? I tried that before but I'm not allowed to do that on the cluster. I can load a cutadapt module but I was hoping making it an executable would solve the problem.

[fkrueger]

Hmm, maybe you need to contact your sys-admins then, you obviously need to get Cutadapt to be working before you can start using it. You should even be able to install it in your home directory and then point there?

[dpryan]

Just install it as a user (pip install --user cutadapt) or use virtualenv.

[padmoo]

I've installed it and when I run felix's command I get:
cutadapt version 1.8.1
...

But I still get this error:
Path to Cutadapt set as: '/gpfs/scratch/cbh12wsu/trim/cutadapt/cutadapt/scripts/cutadapt' (user defined)
1.8.1
Cutadapt seems to be working fine (tested command '/gpfs/scratch/cbh12wsu/trim/cutadapt/cutadapt/scripts/cutadapt --version')
Failed to write to file '1_L.fastq_trimming_report.txt': No such file or directory

Used this command:
bsub -N -q short -o cut3.txt "/gpfs/scratch/cbh12wsu/trim/trim_galore -phred33 --illumina --paired -phred33 --path_to_cutadapt /gpfs/scratch/cbh12wsu/trim/cutadapt/cutadapt/scripts/cutadapt -o adaptrim.fastq -length 50 1_L.fastq 2_R.fastq"

More ideas?

[fkrueger]

Ok, Cutadapt seems to be working now. Do you have write permission in the folder where you keep your FastQ files?

[padmoo]

these are the folder permissions: drwxr-xr-x
I also just did a fastqc run and that adds the files to the folder, so it should be fine.

could it be a problem that the to write file is called .fastqxx.txt?

[fkrueger]

no that shouldn't matter... Could it be that your queuing system does not take you to the same directory (option like -cwd)? What happens if you just run the command without using the queue?

By the way you can shorten the command like this:

/gpfs/scratch/cbh12wsu/trim/trim_galore --paired --path_to_cutadapt /gpfs/scratch/cbh12wsu/trim/cutadapt/cutadapt/scripts/cutadapt [COLOR="Red"]-o adaptrim.fastq[/COLOR] -length 50 1_L.fastq 2_R.fastq

By the way -o is meant to be an output directory, not a file name. This might be the reason why it can't create the output files in the directory called adaptrim.fastq

[padmoo]

It is still running!!!!! What a stupid mistake! Thanks for finding it and shortening my command!

[fkrueger]

Great, enjoy!