Trimmomatic adapter clipping removes many reads even if "keepBothReads" parameter is selected

Question

[al-ash]

I'm using Trimmomatic/0.33 to proces my raw Illumina reads. I've seen some adapter contamination with FASTQC so I first run Trimmomatic as follows:

java -jar trimmomatic-0.33.jar PE R1.fastq.gz R2.fastq.gz PR1.fastq.gz UPR1.fastq.gz PR2.fastq.gz UPR2.fastq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:1:keepBothReads

TruSeq3-PE-2.fa contains:

>PrefixPE/1
TACACTCTTTCCCTACACGACGCTCTTCCGATCT
>PrefixPE/2
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
>PE1
TACACTCTTTCCCTACACGACGCTCTTCCGATCT
>PE1_rc
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA
>PE2
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
>PE2_rc
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

Trimmomatic reports back:

Input Read Pairs: 39618168 
Both Surviving: 28128581 (71.00%) 
Forward Only Surviving: 8927582 (22.53%) 
Reverse Only Surviving: 47164 (0.12%) 
Dropped: 2514841 (6.35%)

I was surprised that despite selecting the option to keep both reads even if they are palindromic via "keepBothReads" there is only 71% of "Both Surviving" : what might be the reads which did not survive? I can think of them only as entirely consisting of adapter sequences - is that correct? Thanks for any suggestion!

Answer 1

[annsun89]

Hi, I read the manual and <keepbothreads> is the name of the parameter but it's not how to set it. Instead, try

java -jar trimmomatic-0.33.jar PE R1.fastq.gz R2.fastq.gz PR1.fastq.gz UPR1.fastq.gz PR2.fastq.gz UPR2.fastq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:1:TRUE