Just a side-note @slh277 here. I noticed that your truncating parameters are very lenient considering the quality graphs you showed. Your truncating point of 250 might be ok for your forward reads but the reverse reads look like they would suffer greatly from that. They look like they drop sharply in quality around 120. I’m not entirely sure of how DADA2 would handle this poor quality set but with my experience of playing around with poor quality paired ends, it doesn’t lead to useful results. It may even lead to low, poor, or not at all joining of the paired ends? Way better to just use the forward reads. Someone with better understanding of that can comment on that!