I have some PE RNAseq reads contaminated with adapter sequences.
Before removing the adapters, i mapped the reads using bowtie2 -> samtools -> picard-tools "CollectInsertSizeMetrics.jar" and got histogram as shown in the first figure.
After removing the adapters with Trimmomatic, i again run bowtie2 -> samtools -> "CollectInsertSizeMetrics.jar" and got the 2nd histogram with many RF reads, as shown in blue color in the plot. Actually here i already set bowtie2 parameter --no-discordant to remove discordant reads.
I cannot figure out the reason there are so many reads flip their orientations from FR -> RF? I wonder if this change are caused by Trimmomatic, or picard? How this happened?! Any one can help?