Question: Hi all,

Usually get on well with Trimmomatic but am having issues removing adapters from some paired-end RNA-seq data. Here's my process so far:

1) Put together a PE adapter fasta for the adapters described by the sequencing company; ran trimmomatic; no reads dropped.

2) Ran Trim_galore! to automatically detect adapters. It detected the Illumina sequence `AGATCGGAAGAGC` in ~30% of reads.

3) I grepped for this sequence in the raw FASTQs (and used flag `-B 1` to also retrieve the metadata line). Sure enough, I could see this sequence in the grepped fastq entries, plus 20 bp following that were different for Read 1's vs Read 2's. (Images 1 and 3 respectively, see highlighted regions for an example.)

4) I searched the Illumina adapters PDF for these sequences, and they turn out to be a perfect match for the "IDT for Illumina TruSeq DNA and RNA UD Indexes" (see image 2). I then confirmed with the sequencing company that this was what they had in fact used - yes, the ones they initially sent me were wrong. Great, back to trimmomatic then!

5) Ran trimmomatic looking for these adapters (which I had confirmed with my own eyes were present in at least a handful of reads - hopefully you can confirm this for yourself using the screenshots provided). Zero reads dropped. I tried every combination of the adapter sequences I could think of, including complements with non-complements, read 2 adapters taking the place of read 1 adapters, etc, etc. Still zero reads dropped. (See below for a list of all pairs of adapter sequences I submitted to trimmomatic.)

What am I doing wrong? I will likely just accept the results of trim_galore and move on, but this is driving me crazy as I can't see any reason why it isn't working.

ADAPTER SEQUENCES TRIALLED:
(formatting being mucked around by BioStars, the "Prefix.../1" was in fact on a separate line to the nucleotides and there were angle brackets (greater-than-signs) at the start of each "Prefix..." line.)

>PrefixIllumina1/1
AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
>PrefixIllumina1/2
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
>PrefixIllumina2/1
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
>PrefixIllumina2/2
AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
>PrefixIllumina3/1
TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGT
>PrefixIllumina3/2
TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA
>PrefixIllumina4/1
TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA
>PrefixIllumina4/2
TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGT
>PrefixIlluminaOne/1
AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
>PrefixIlluminaOne/2
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
>PrefixIlluminaTwo/1
AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
>PrefixIlluminaTwo/2
TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA
>PrefixIlluminaThree/1
TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGT
>PrefixIlluminaThree/2
TCTAGCCTTCTCGCAGCACATCCCTTTCTCACA
>PrefixIlluminaFour/1
TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGT
>PrefixIlluminaFour/2
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT`

![Screenshot of example read 1's][1]
![Illumina adapter PDF screenshot][2]
![Screenshot of example read 2's][3]

[1]: /media/images/55c5783b-8f08-408b-9074-b0d9e60a
[2]: /media/images/315841a2-8364-4929-bf81-4c5c62e4
[3]: /media/images/7d5390dd-a9d4-4306-b04a-0b8f5725

Answer: I think you are overcomplicating things, make a simple adapter file like so:

>adapter
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

then cut with that.

Trimmomatic has some additional features and behaviors called palindromic trimming and I think naming your reads with trailing /1 and /2 triggers that behavior.