Trimming and realigning the paired-end reads in which both 3' ends overlap each other. A junction sequence is shown with the junction site noted by a yellow dot. The blue region is from Gene A, and orange is from Gene B. The paired-end read with overlapped 3' ends (black thick line) cannot map to Gene A and Gene B, as reads cover the junction site. A series of trimmed reads (gray thick line) are obtained by iteratively trimming 5 nucleotides (nts) each time from the 3' ends until the reads could map to Gene A and Gene B. In this example, end 1 of a paired-end read requires two cycles of trimming to achieve successful alignment, while end 2 needs five cycles.