Thanks for clarifying, @Hui_Yang!
515F 926R is ~411 nts long. Based on the provenance in the files you shared, your trim and trunc params were:
- trunc_len_f: 240
- trim_left_f: 20
That means that the forward reads were trimmed to 220 nts
- trunc_len_r: 200
- trim_left_r: 20
That means the reverse reads were trimmed to 180 nts
I think that means there is no way the fwd and rev reads can overlap:
411 nt amplicon |----------------------------------------------|
240 nt fwd read |-------------------------->
200 nt rev read <---------------------------|
29 nt overlap ^^^^^^^^^
@SoilRotifer, am I doing this math right?
I think you will need to relax your params quite a bit, you need at least 23 nts more to ensure sufficient overlap.