[felvis56]

Thank you for the fast reply.

Apologies if I am being basic. If there is a single base with Q<25 but the following bases are ok will the read be cut at that point or is there a set number of bases needed to be below Q<20 resulting in the read being cut?

When I look at my fastq files on FastQC the error bars do dip below 20 but I was thinking this was due to a small number of bases over multiple reads.

Thanks

[fkrueger]

Hi Fiona,

quality trimming removes the portion of the read where the qualities become minimal, but does not remove then entire read (pair) completely. This is taken from Cutadapt --help:

 --quality-base=QUALITY_BASE
                        Assume that quality values are encoded as
                        ascii(quality + QUALITY_BASE). The default (33) is
                        usually correct, except for reads produced by some
                        versions of the Illumina pipeline, where this should
                        be set to 64. (Default: 33)

[felvis56]

I wanted to ask a question about the quality trimming of sequences. I have Illumina reads and use Trim Galore to remove adapters and primers with good success.

Is the quality trimming based on the average Phred score of the read or if I use a cutoff of 25 and have any/some bases below 25, will the whole read be removed?

Thanks,

Fiona

[fkrueger]

Great, enjoy!

[padmoo]

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

[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]

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]

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

[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?

[dpryan]

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

[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?

[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]

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]

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]

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

?