PICRUST2 removing all ASV sequence

Dear Qiime 2 community,

I'm trying to do pathway analysis using PICRUST2. All files are changed to the required format which is .fna and biom. However, when I apply this command " place_seqs.py -s seqtablefungi.fna -o out.tre -p 2" all the sequences are removed and only one is retained for downstream analysis. How can I solve this problem? The output message says "This is the set of poorly aligned input sequences".

I would really appreciate your assistance. here is there image of my codes and output.

Good afternoon,

The reason is hiding in the warning message: :mag:
Warning - 1448 input sequences aligned poorly to the reference sequences. ... will be excluded from downstream steps.

As also mentioned in the error, consider adjusting the --min_align option.

Also check that your ASVs are in the same direction as the reference, as reverse and reverse complemented orientations are not considered! :repeat:

Hi Colin, thanks for the response. Do you think if I use the code min_align, it will also include the sequences that are poorly aligned?

How do I check if ASVs are in the same direction with reference?

Yes. You could pass --min_align 0.7 to drop the inclusion threshold to 70%, or --min_align 0.6 to drop the inclusion threshold to 60% similar to reference.

However, it's pretty strange for almost none of your reads to be matching against the references. This is why we should check if something else is going on.

There's a few ways I've done this before, but I'm not sure what's the recomended pipeline with Qiime2.

Thanks so much Colin, could you kindly share the ways you know and the type of pipeline? Because I used DADA2 to generate these ASVs.

Thank you!!