Trimmomatic: Not trimming..

[lchippy]

Hey! I am trying to use Trimmomatic to trim Illumina adapters sequences from my TruSeq Small RNA library SE 75bp reads off of a HiSeq but I am unsuccessful... I'm fairly novice so I am probably making a novice mistake and would appreciate any help!

Here is the script I am trying to run:

java -jar ~/applications/Trimmomatic-0.36/trimmomatic-0.36.jar SE -threads 4 -phred33 -trimlog ~/path/sample1_trimlog ~/pathtoinput/sample1.fastq ~/pathtooutput/sample1_trim.fastq ILLUMINACLIP:~/applications/Trimmomatic-0.36/adapters/smRNA_TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:10

The smRNA_TruSes3-SE.fa:
>TruSeq3_IndexedAdapter
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
>TruSeq3_UniversalAdapter
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA
>RPI2
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
>RPI9
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG
>RPI10
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG
>RPI11
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG
>RPI4
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG
>RPI5
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
>RPI6
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG
>RPI7
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG

Thanks for any help!

[mastal]

You have 'PE' in your command, but you appear to be trying to trim SE data, because you are providing the names of one input file and one output file only, so you should change that to 'SE'.

[lchippy]

Oh shoot, that was troubleshooting. I was running this command with the SE designation but since I was having problems I started marching along the command and changing each part just to see if that helps. (Ie/ i’ve Done it with SE and it hasn’t worked)

Does my ILUMINACLIP part look correct?

[mastal]

Your command looks OK, why do you think it's not working?

Is trimmomatic running, and what output do you get when it finishes?

[lchippy]

Trimmomatic runs and seems to do everything except trim Illumina adapter sequences. Ie/ It runs normals, trim low quality bases, doesn't fail, and takes what seems to be a appropriate about of time. It also outputs a "trimmed" file though the illumina adapters are still there... At first I thought it was my adapter files, so on top of using a file I created, I also downloaded one I found online. (https://github.com/Transipedia/dekup...er/adapters.fa)

So not sure whats wrong but happy for any troubleshooting tips!

[mastal]

Have you tried running trimmomatic with only the ILLUMINACLIP command and not the rest of the quality trimming, just to see how the Illuminaclip part works?

Are you sure you are using the right adapter sequences/barcodes that were used with your samples?

[kmcarr]

Originally Posted by lchippy

Hey! I am trying to use Trimmomatic to trim Illumina adapters sequences from my TruSeq Small RNA library SE 75bp reads off of a HiSeq but I am unsuccessful... I'm fairly novice so I am probably making a novice mistake and would appreciate any help!

Here is the script I am trying to run:

java -jar ~/applications/Trimmomatic-0.36/trimmomatic-0.36.jar SE -threads 4 -phred33 -trimlog ~/path/sample1_trimlog ~/pathtoinput/sample1.fastq ~/pathtooutput/sample1_trim.fastq ILLUMINACLIP:~/applications/Trimmomatic-0.36/adapters/smRNA_TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:10

The smRNA_TruSes3-SE.fa:
>TruSeq3_IndexedAdapter
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
>TruSeq3_UniversalAdapter
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA
>RPI2
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
>RPI9
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG
>RPI10
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG
>RPI11
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG
>RPI4
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG
>RPI5
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
>RPI6
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG
>RPI7
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG

Thanks for any help!

You have the wrong sequences in your adapter file. The TruSeq Small RNA adapter sequences differ from the standard TruSeq RNA/DNA adapters. Also, you do not need the individual index sequences, just the common portions of the Index and Universal adapters are needed. Here is the TruSeq Small RNA file I use with Trimmomatic:

>TruSeq3_smRNA_IndexAdapter
TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC
>TruSeq3_smRNA_Universal
GATCGTCGGACTGTAGAACTCTGAACGTGTAGA