Sorry about the delay in response here. Long weekend up in , been away from the computer most of it.
So when you set everything to default and the truncating parameter to 50, 100, or 150 bp you get 0 reads passing?
--p-trunc-q 250 will have the opposite effect of what you are expecting.
--p-trunc-q INTEGER Reads are truncated at the first instance of a
quality score less than or equal to this value. If
the resulting read is then shorter than `trunc-len`,
it is discarded. [default: 2]
Meaning that all of your reads will be truncated at the very first position because any number will be below 250, thus will be filtered out. It also doesn’t really make sense to set it that high since q-scores here will have a max of 40 anyways, so no surprise that no reads passed in those runs. Those parameters are not your issues here, there is something else causing all of your reads to be filtered out.
In this case if you trimmed 40 from the 5’ and truncated at the 55 position from the 3’, that means your reads were only 15 bp in length and DADA2 has a minimum length retention of 20 bp reads by default so again it is no surprise all of those reads were also discarded.
A couple of suggestions:
Have you removed your primers/adapters from these sequences? Those tend to cause some problems with dada2, especially if they were degenerate primers. Make sure this is done and try my initial trim/trunc suggestion with default settings.
Try setting your trim parameter to 30 and your truncating value at say 130, with maxee to 6 to start. This is just to give us an idea where the issue is coming from. If nothing is still passing then we may have a bit more serious issue.
There’s also the possibility that your reads have ambiguous
N nucleotide in them which will cause dada2 to discard that whole read. Have a look through some of your raw reads to see if that may be the case.
Let’s try those first and go from there.