Hello all,
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
--i-demultiplexed-seqs Baywide_microbiome/all_samples/Baywide-full-demux-paired-end.qza
--p-trim-left-f 18
--p-trim-left-r 18
--p-trunc-len-f 290
--p-trunc-len-r 200
--p-n-threads 48
--o-representative-sequences Baywide_microbiome/all_samples/trim290f-200r/full-rep-seqs-dada2-trim290f-200r-18left.qza
--o-table Baywide_microbiome/all_samples/trim290f-200r/full-table-dada2-trim290f-200r-18left.qza
--o-denoising-stats Baywide_microbiome/all_samples/trim290f-200r/full-stats-dada2-trim290f-200r-18left.qza
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
--i-demultiplexed-seqs Baywide_microbiome/all_samples/only_forward/Baywide-full-demux-single-end.qza
--p-trim-left 18
--p-trunc-len 290
--p-n-threads 48
--o-representative-sequences Baywide_microbiome/all_samples/only_forward/rep-seqs-dada2-trim18-290.qza
--o-table Baywide_microbiome/all_samples/only_forward/table-dada2-trim18-290.qza
--o-denoising-stats Baywide_microbiome/all_samples/only_forward/stats-dada2-trim18-290.qza
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).
Thanks everyone!
Baywide-full-demux-paired-end.qzv (322.9 KB) full-stats-dada2-trim290f-200r-18left.qzv (1.2 MB) Baywide-full-demux-single-end.qzv (296.7 KB) stats-dada2-trim18-290.qzv (1.2 MB)