Hello Armin,
Thank you for posting this really great question along with the detailed screenshots. I think you are on the right track.
denoised: 99,046, merged: 57
Yep, that's your problem. Let's fix it!
Sure. The pair of Illumina reads sequence your amplicons from both ends, with some overlap in the middle.
250 bp amplicon |-------------------------|
150 bp read |--------------->
150 bp read <---------------|
50 bp overlap (correct!) ^^^^^
Here's how I work it out:
(forward read) + (reverse read) - (length of amplicon) = overlap
150 forward + 150 reverse - 250 amplicon = 50 overlap
This matches the diagram I showed above.
For your data, here are the numbers I know so far:
216 forward + (218 - 2) reverse - (16S-341F and 16S-805R) amplicon = overlap
216 forward + 216 reverse - (805 - 341) amplicon = overlap
432 - 464 = overlap
-32 = overlap
EDIT: So your overlap is negative, which means there is a 32 bp gap between your paired ends.
I agree! Let's me know if this helps answer your questions, then we can see how we can fix it!
Colin