I know this has been posted about several times and I have gone through many forum posts with the same issues, but I was hoping someone might be able to help me out on this. Any help would be very much appreciated.
Basically, my issue is low %filtered and %merged about running dada2. I'm looking at V3V4 regions, so I think I understand that my sequences should at least be (805-341)+20 = 484bps.
So to begin with, the quality scores of my reverse reads are not great:
But I decided to plow on with paired denoising because as someone pointed out, sequencing is expensive, so let's try to use everything I have first. These were my trimming and truncating parameters:
qiime dada2 denoise-paired
Which I realise fails the overlapping required since 290-18+200-18 = 454. Which might explain my %merging issue. But I don't think I can lower the truncation any more because of my not great reverse reads. Here's a screenshot of my dada2-stats:
I've sort from the lowest, but basically my %filter range is from 0 - 71.27%. I wonder about the first four samples, which had very little input to begin with. But even discounting them, my %filter range is from 52.58 - 71.27%.
So I thought, ok fine, let's just try the forward reads then. I used basically the same parameters because I figured the forward read looks pretty good.
qiime dada2 denoise-single
However, that didn't really solve my %filtered problem, with it still ranging from 19.17 - 77.75%. I guess the bottom range has improved, but why not the top range?
I am pretty new to all this and have been consulting a more experience user about all this as well. But figured extra eyes will definitely help as well.
I am running QIIME 2021.2.0 on Linux (Ubuntu).