Hi everyone,
I am blocked on my quality filtering part of analysis and I need some help.
I used dada2 denoising on my microbiome analysis and it seems there are many reads filtered out on the way. In my opinion too many. I have searched through forum and tried to refer what was posted to my case.
https://forum.qiime2.org/t/dada2-denoise-paired-removing-most-sequences-during-filtering/7159
https://forum.qiime2.org/t/dada2-denoise-paired-result-90-loss-in-reads/619
I worry it affects microbial community.
Here is what I did:
Sequencing 2x250, V3-V4 region, Illumina Miseq
I followed moving pictures tutorial.
Commands:
qiime dada2 denoise-paired
--i-demultiplexed-seqs FastQ-demux.qza
--p-trunc-q 20 //I did the same denoising part having only 10 value here, for checking what went wrong
--p-trim-left-f 8
--p-trim-left-r 8
--p-trunc-len-f 240
--p-trunc-len-r 240
--p-n-threads 0
--p-chimera-method consensus
--o-representative-sequences rep-seqs-dada2-chill10.qza
--o-table table-dada2-chill10.qza
--o-denoising-stats stats-dada2_chill10.qza
FastQ-LB18_31-demux.qzv (290.6 KB)
--p-trunc-q 20
stats-dada2_LB18_31.qzv (1.2 MB)
--p-trunc-q 10
stats-dada2_LB18_31-chill10.qzv (1.2 MB)
Are my parameters too strict?
In my opinion problem may be merging (based on --p-trunc-q 10) but I dont know how to find the reason for it.
Based on --p-trunc-q 20 results too many read are filtered out in the first instance.
It is pretty confusing.
Regards,
Joanna