Hello,

I'm using the ILLUMINACLIP step in Trimmomatic to remove adapter and index sequences from paired-end Illumina RNAseq data.

I am generating four output files; paired read 1 and paired read 2, and unpaired read 1 and unpaired read 2. I have run fastqc on all four output files. For the paired data, all adapter and index sequences ('overrepresented sequences') have been removed, but for the unpaired reads many contaminated reads remain. I attach the fastqc output for unpaired read 2.

I have screened for the adapter sequence and the reverse compliment of this (which makes up the bulk of the overrepresented reads in the attached fastqc report) using both simple and palindrome trimming, and tried altering the mismatch and threshold parameters, but always getting a similar result to that attached.

Here is the command I have used:

java -classpath trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticPE -phred33 NG-6077_14E_lib11117_1_sequence.fastq.gz NG-6077_14E_lib11117_2_sequence.fastq.gz 14E_1_paired.fastq 14E_1_unpaired.fastq 14E_2_paired.fastq 14E_2_unpaired.fastq ILLUMINACLIP:truseq_adapters_pl.fa:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:25

Can anyone suggest a fix for this?

(sorry this is in MapMan - I couldn't add a new post in the Robin category ...)

Thanks.