if the adapter.fa is needed and when to set keepbothreads TRUE

[taryl]

Hi.

I used to Trimmomatic to trim my bulk RNA sequencing data (pair-end). Adapters were pre-removed in some of my data by the company.
I tried to use timmomatic to do the trimming and I have doubts that: 1) if the adapter.fa was needed and 2) when should I set keepbothreads TRUE.

Q1:
For those Sample_RNA_seq.fq.gz file which have been removed adapters by the company, should I used the “ILLUMINACLIP” parameter like:
Trimmomatic PE -phred33 Sample_RNA_seq_1.fq.gz Sample_RNA_seq_2.fq.gz Sample_RNA_seq _trimmo_paired_1.fq.gz Sample_RNA_seq _trimmo_unpaired_1.fq.gz Sample_RNA_seq _trimmo_paired_2.fq.gz Sample_RNA_seq _trimmo_unpaired_2.fq.gz ILLUMINACLIP: TruSeq2-PE.fa:2:30:10 SLIDINGWINDOW:4:15 LEADING:3 TRAILING:3 MINLEN:50

OR should I drop the parameter like:
Trimmomatic PE -phred33 Sample_RNA_seq_1.fq.gz Sample_RNA_seq_2.fq.gz Sample_RNA_seq _trimmo_paired_1.fq.gz Sample_RNA_seq _trimmo_unpaired_1.fq.gz Sample_RNA_seq _trimmo_paired_2.fq.gz Sample_RNA_seq _trimmo_unpaired_2.fq.gz SLIDINGWINDOW:4:15 LEADING:3 TRAILING:3 MINLEN:50

Q2:
I didn’t understand when to set keepbothreads TRUE. For the default False, my previous understanding was that if the two reads (forward read Sample_RNA_seq_1.fq.gz in and the reverse read in Sample_RNA_seq_2.fq.gz) are in reverse complement, the corresponding reverse read in Sample_RNA_seq _trimmo_paired_2.fq.gz will be dropped, resulting some reads without paired one in Sample_RNA_seq _trimmo_paired_2.fq.gz. However, I tried to trim the data by setting keepbothreads False, the total reads number were same in Sample_RNA_seq _trimmo_paired_1.fq.gz and Sample_RNA_seq _trimmo_paired_2.fq.gz.so I think I have misunderstood.

Thanks for your time and concern!