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Old 11-28-2014, 05:51 AM   #1
dena.dinesh
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Default Trimming multiple adapters in a single run

Hi,

I ran the quality control for my fastq reads and found that some adapter seuqences in every sample. I used Fastx_clipper like this:
fastx_clipper -a "AGTCGTATAGCAGT" -v -i /input/fastq/file -o /trimmed/output/file.

How can I trim the fastq file with multiple adapter.. i have one more adapter sequnces which has to be trimmed.

How can I use multiple adapters in a single run?

Kindly Guide me.
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Old 11-28-2014, 06:07 AM   #2
blancha
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As far as I know, you can only trim one adapter at a time with fastqx_clipper or cutadapt.
You'll have to trim them one after the other if you have different adapters for the reads.

Note that, with Cutadapt at least, you only need to trim the first adapter after the insert.
This sequence is generally identical for all the reads. It doesn't matter if the sequences differ after the first adapter.

With Trimmomatic, you can give in argument an adapter file with all the adapter sequences.
http://www.usadellab.org/cms/?page=trimmomatic
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Old 11-28-2014, 06:13 AM   #3
dena.dinesh
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You mean to say first trim the raw sequence with first adapter output it. Then later trim the outputted file from the first trim to trim the next adapter..Am I right?
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Old 11-28-2014, 06:23 AM   #4
blancha
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No.
You'll have to check the behaviour of your trimmer.
In the case of Cutadapt, you just need to trim the first adapter.

You don't need to know all the adapter sequences. You just need to know and trim the sequence of the first adapter after the insert.

E.g. The adapter sequence is TAAC

Before trimming:
ATCATAACAT
CTAGTAACTT

After trimming:
ATCA
CTAG
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Old 11-28-2014, 06:35 AM   #5
dena.dinesh
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Hi,

Like you said I had two adapter sequences before I trimmed. First I trimmed using first adapter sequence using fastx_clipper and later uploaded the sequence in Fastqc to re-check, surprisingly the second adapter sequence which shown before was also removed and now I have nor adapter sequences...But how come my second adapter disappeared..Can i take this trimmed fiel for further analysis?
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Old 11-28-2014, 06:40 AM   #6
blancha
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Yes. The trimmer will cut off the rest of the sequence once it encounters the adapter.
Hence, you only need to trim the first adapter after the insert.
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Old 11-28-2014, 06:42 AM   #7
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That's the default behaviour for Cutadapt anyway.
I believe that the other trimmers use the same algorithm, but cannot absolutely guarantee it.
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Old 11-28-2014, 06:44 AM   #8
dena.dinesh
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Also Is it necessary to filter reads based on quality score when you find quality score for all bases is above 25 or more.?
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Old 11-28-2014, 06:53 AM   #9
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It depends on the type of experiment.

I wouldn't trim for RNA-Seq experiments measuring gene expression levels, or ChIP-Seq experiments measuring immunoprecipitated DNA.
I might trim at 30 or 32 for experiments whose objective is to find mutations (whole-genome seq, exome-seq and sometimes RNA-Seq) or methylation levels (bis-Seq).

For RNA-Seq experiment or ChIP-Seq experiments, trimming would do more harm than good if all your bases are above 25.
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Old 11-28-2014, 10:21 AM   #10
luc
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In my eyes fastqx_clipper is pretty outdated by now. Especially BBduk, but also Trimmomatic, and Cutadapt, have been developed further and offer way more options.
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