Hi,

I have some questions concerning the output of Trimmomatic after adapter removal.
I have 80 bp paired-end reads in Ilumina 1.9 encoding (Phred+33). Using FastQC for quality control, I noticed some overrepresented sequences in the data which were identified as TruSeq adapters. For this reason, I used Trimmomatic in order to trim the adapters and to drop any resulting reads with a length < 36 bp:

    java -jar trimmomatic-0.38.jar PE -phred33 seq_1.fastq.gz seq_2.fastq.gz seq_1_trimmed_paired.fastq.gz seq_2_trimmed_unpaired.fastq.gz seq_1_trimmed_paired.fastq.gz seq_2_trimmed_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 MINLEN:36

As a result, I get ~99% of both reads surviving and ~1% forward reads only surviving and 0% reverse reads only surviving:

    ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
    Input Read Pairs: 71446282 Both Surviving: 70983555 (99.35%) Forward Only Surviving: 453784 (0.64%) Reverse Only Surviving: 0 (0.00%) Dropped: 8943 (0.01%)

Is the 0% reverse only surviving the expected result?
It seems that the reverse reads are the only ones affected by the adapter trimming. However, in the FastQC quality control, the warnings for overrepresented adapter sequences only showed up for the forward reads.

And what is the difference between the TruSeq3-PE.fa and the reverse complements TruSeq3-PE-2.fa adapter sequence files and which of them should actually be used to trim adapters from paired-end reads?

I would be very grateful for any help or explanations.
Trimmomatic's default behaviour is to drop the reverse reads when it trims adapters, so you get forward reads only surviving as a result. 

The reasoning behind this is that when you read into the adapter sequences it means that the insert is shorter than one of the reads, so the reverse read doesn't add any extra information, it is just the reverse complement of the forward read. 

However, the default behaviour can be changed if you want to keep the reverse reads after adapter trimming. See the Trimmomatic manual, you need to add TRUE as the last parameter to ILLUMINACLIP.

