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Old 05-16-2020, 01:50 PM   #1
iramai
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Location: Eibar

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Default How to trim dual index adapters with Trim Galore!

Hi everybody,
I am trying to trim some adapters with Trim Galore from my bisulfite sequencing data, but I don't know what command to use.
It has been really difficult to take the information of library construction and I only know that it has been done using IDT unique dual indexing adapters in paired end data, detailed as follows:

1 - P5 ATATGCGC, P7 CTGATCGT
30 - P5 AACGTCTG, P7 GCGTCATT
31 - P5 TATCGGTC, P7 ATCCGGTA
32 - P5 CGCTCTAT, P7 CGTTGCAA

When doing the FASTQC analysis I have found some overrepresented sequences such as:
- In one of the sequence of the pair
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
- In the other sequence of the pair
GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

An also in the adapter content section the Illumina Universal adapters are identified

Does all this make sense?
So I need to add something more to those P5 and P7 adapter sequences when running Trim Galore?

Will this command be correct for the first example of unique dual index adapters?

> trim_galore --paired -a ATATGCGC -a2 CTGATCGT -q 15 --stringency 5 -e 0.05 --length 50 --fastqc --gzip file_1.fastq.gz file_2.fastq.gz

Thanks in advance!
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Old 05-16-2020, 02:50 PM   #2
GenoMax
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Index sequences are never part of an actual read in Illumina sequencing so you are never going to find index sequences you noted above in R1/R2 reads.

As for the poly-A and ploy-G (no signal, two color chemistry) you just need to remove them during trimming.
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Old 05-17-2020, 08:04 AM   #3
iramai
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Thanks for your answer!
As the only information of the sequencing service was that about the dual index adapters I though that those were the sequences that I need to trim.
So for the command how do I need to add this poly A, T and Gs?
One by one in each sequence file? or combining the paired end sequences somehow?

Thanks again!
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Old 05-17-2020, 10:39 AM   #4
GenoMax
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I suggest you try "bbduk.sh" from BBMap suite instead. A guide is available. You can trim out all these things in one pass.

I am not a trim galore user but you may be able to additional specify poly-A and poly-G there as well.
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