I have a quick question in regard to truncating parameters for ITS2 (Its4Fun, 5.8s primers). I have looked through the tutorial, the forum and even went on google to see if I could find some help but I am still at a lost. I did try to install ITSx to test it out, but I keep getting an error (I can go into this in more detail if you guys suggest I should choose this method, as opposed to DADA2).
I am working with fungal data, using the primers stated above, and after playing around w the parameters for DADA2 (mostly the truncating parameter), I cannot seem to get over a 60% output from DADA2.
What I have tried:
I have ran over ~20 different parameters so far.
If I choose smaller more stringent values for truncating <185, I get a high number of sequencing passing the filtering (70-80%) step but they all fall off at the merging step (end up with about 35-45%).
Fungal-Stats-dada2-171-165.qzv (1.2 MB)
Too high number results in similar results as #3
The number that seems to work the best is 209-190 but I end up with ~60% passing the filtering step and an overall of ~58 after merging.
I have tested various truncating parameters and I cannot manage to get over 60% after merging. Fungal-Stats-dada2-205-190.qzv (1.2 MB)
So I am a little stuck, is this good enough to proceed? If not, what am I doing wrong or how can I retain more sequences when processing through DADA2?
Here is my demux file. Fungal-demux-trimmed.qzv (298.6 KB)
For my code, I have also added the “–q-trunc” parameter as previously suggested, but whether I set it to 0 and or 2, it doesn’t seem to make a difference.
If you could give me some advice, I would really appreciate it.