Hello everyone, am trying to analyse 16S Pacbio sequence from a chicken experiment and am getting very low input non chimera percentage. Can someone help me? The output is attached
dada2-ccs_stats.qzv (1.2 MB)
Hi @oluwatobi,
Can you please share the command you used to get this output so I can see what parameters you are using?
--Hannah
Thank you for the reply.
qiime dada2 denoise-ccs --i-demultiplexed-seqs ./tobi.qza
--o-table dada2-ccs_table.qza
--o-representative-sequences dada2-ccs_rep.qza
--o-denoising-stats dada2-ccs_stats.qza
--p-min-len 1000 --p-max-len 1600
--p-front AGRGTTYGATYMTGGCTCAG --p-adapter RGYTACCTTGTTACGACTT
--p-n-threads 4
#######
Summarizing denoised statistics
qiime metadata tabulate --m-input-file ./dada2-ccs_stats.qza
--o-visualization ./dada2-ccs_stats.qzv
Hi @oluwatobi,
Can you also send me your demux.qzv. I am suspecting that your issue is not the chimeric filter but the denoising and filtering steps before the chimeras are filtered out.
--Hannah
tobi.demux.summary.qzv (314.2 KB)
Hi @oluwatobi,
After looking at your demux there is definitely something going wrong with how the pacbio sequences are being imported. I found this (old but still helpful) post about pac bio encoding regarding what is happening with your quality scores.
I have created an issue with our development team to get this problem addressed but until it is fixed there is not much to do inside the qiime2 ecosystem but wait. I wish I had better news but hopefully it gets fixed soon!
If you are comfortable with trying some third party options I found this forum post regarding a work around for this. Keep in mind you will not have provenance for this step.
I hope this is helpful!
--Hannah
Thanks for your reply, I will look into these options while I await solutions from your guys.
Any updates on this, or could you work me through the second step of the solution you provided?
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