Got some 101 bp paired end illumina reads I recently got back and I'm in the middle of QC. I decided to use Trimmomatic over solexaqa which I normally use. Problem is, my paired 'cleaned' reads outputted from trimmomatic are uneven file sizes and reads. I have used the option with ILLUMINACLIP , keepbothreads ( ":TRUE"). I decided this was best as to my knowledge the Trinity de novo assembler needs paired end data to work best.
The trimmomatic specific code I run (as part of a loop):
java -jar directory/trimmomatic-0.35.jar PE -phred33 -basein $f1 -baseout "$f1"filtered.fastq ILLUMINACLIP: directory/adapters/TruSeq3-PE-2.fa:2:30:10:8:TRUE HEADCLIP:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Trimmomatic runs as normal, and produces all files. Can I also ask a few ignorant questions. I am correct in assuming headclip removes reads from the 5'? Additionally, after the trimming has succeeded I still have a flagged high kmer content at the start of the 5' where hexamer primer bias usually exists. Any suggestions or just an artifact to ignore?
I am using TruSeq adaptors.I am assuming all sequencing facilities will now work with TruSeq3?
Thanks for helping.