Trimmomatic IlluminaClip

[hamcan]

Hi everyone,

I ran a 2x250bp HiSeq run on water samples to analyze what species of bacteria are present. So the whole aim of my project is to look at the species abundance and the functional analysis.

I had my adapter sequences present in some of my reads and therefore I decided to use trimmomatic to both trim and remove my adapter sequences. I used the following parametrs and wanted to know if they were good. My adapter sequence is 33bp in length (fastqc said I only had nextera transposase sequence contamination). Note that I used the nextera adapter file that trimmomatic provided:

java -jar trimmomatic-0.36.jar PE 103_S80_L001_R1_001.fastq 103_S80_L001_R2_001.fastq MP103_R1paired.fastq MP103_R1unpaired.fastq MP103_R2paired.fastq MP103_R2unpaired.fastq ILLUMINACLIP:Adapters.FASTA:2:30:10:4 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:25 MINLEN:36

Once I did the command at the top, I did a FASTQC quality report check and I received the following:

I failed the Per base sequence content and the per base sequence GC content. I got ! on Kmer content and sequence length distribution.
See images below. Essentially, what parameters should I be more worried about passing and failing?
And are my trimmoatic illumina clip parameters good?

Thanks in advance!

[GenoMax]

This question has been asked countless times and the answer is the same. Having a red "X" on a FastQC test does not automatically make the data bad. Those limits are configurable and are generally set for plain genomic DNA sequencing.

Take a look at Dr. Simon Andrew's blog posts that address some of the common questions at this link. They should answer some of your questions.