Cutadapt - help.

[vishwesh]

Hi,
I am using cutadapt for removing the adapter sequence. I have 2 adapter sequence.

RNA 5Adapter (RA5)
5 GUUCAGAGUUCUACAGUCCGACGAUC
RNA 3?Adapter (RA3)
5 TGGAATTCTCGGGTGCCAAGG

The 1st one is 5' adapter and 2nd is 3' adapter.

I am using the following command line to remove the adapter seq.

cutadapt -a TGGAATTCTCGGGTGCCAAGG -g GUUCAGAGUUCUACAGUCCGACGAUC input.fastq > output.fastq

Length Distribution I get
Mean sequence length: 32.49 ± 10.53 bp
Minimum length: 16 bp
Maximum length: 51 bp
Length range: 36 bp
Mode length: 51 bp with 2,852,626 sequences


And I found that the 5' adapter has U instead of T. Will that be fine?

I tried replacing U with T GUUCAGAGUUCUACAGUCCGACGAUC > GTTCAGAGTTCTACAGTCCGACGATC and tried removing adapter sequence.

cutadapt -a TGGAATTCTCGGGTGCCAAGG -g GTTCAGAGTTCTACAGTCCGACGATC input.fastq > output.fastq

Length Distribution I get
Mean sequence length: 31.26 ± 11.29 bp
Minimum length: 1 bp
Maximum length: 51 bp
Length range: 51 bp
Mode length: 51 bp with 2,805,271 sequences

I get varied length distribution in both the cases. Which one should I choose..
First is the command that I am using is right??
I am working on small RNA seq data.
Kindly let me know.

Thanks in advance.

Regards
Vishwesh

[Brian Bushnell]

Reads don't come out of sequencers with 'U' in them, just 'T', so don't use 'U' for postprocessing reads. Some tools may understand it but most won't.

And a varied length distribution is expected, is it not?

[vishwesh]

Thanks for the response.
Hope the command that I use is correct.
Can you pls let me know how remove 5' adapter and 3' adapter using cutadapt??

Regards
Vishwesh

[Brian Bushnell]

Well, no, I don't use cutadapt. I can advise you on bbduk, though.

bbduk.sh -Xmx1g in=input.fq out=trimmed3.fq ktrim=r literal=TGGAATTCTCGGGTGCCAAGG k=19 mink=12

This will trim to the right when finding an adapter.

bbduk.sh -Xmx1g in=trimmed3.fq out=trimmed3_5.fq ktrim=l literal=GTTCAGAGTTCTACAGTCCGACGATC k=19 mink=12

This will trim to the left when finding an adapter.

If you want to allow a mismatch, you can set "hdist=1".

-Brian

[relipmoc]

try skewer

skewer is also qualified for trimming adapters from small-RNA reads.

for your case, you may use the following command:

skewer -x TGGAATTCTCGGGTGCCAAGG small-RNA.fastq -1 | skewer -x GUUCAGAGUUCUACAGUCCGACGAUC -m head -r 0 -l 21 -L 25 -o trimmed -