Hi, I am processing my pacbio 16S rRNA reads through denoise-ccs and as stats-dada2.qzv (1.2 MB) you can see from the table I am losing almost 60% of my reads at the denoising step. Could anyone help me to improve this? I have tried changing the --p-max-ee but that does not help.. dada2 denoise-…

@kumars , The go to place to start when you are losing more reads than you expected, is to really double check that all non-biological sequences have been removed from your reads, so that is a good place to start. You also might want to watch these videos from one of our workshops about both the the…

Thanks @Keegan-Evans , I have seen those videos, they are very good and informative. I tried without --p-min-len and --p-max-len which did not change the outcome and I am still losing almost the same amount of reads. I was wondering, If removing these parameters, isn't that set it to default mode? I…

@kumars , Can you tell me more about the wet lab processing that was performed on your samples prior to sequencing? If you do not know this, do you know which PacBio machine your sequences were read on? If no PCR was performed, it might be that DADA2 is seeing true, unique sequences that are very c…

@Keegan-Evans , Prior to sequencing, PCR was performed to amplify ~ full-length 16S rRNA using 27F and 1492R primer pairs. PacBio Sequel II sequencing platform was used. I have tried another DADA2 run with "--min-fold-parent-over-abundance" 1. The outcome is still the same. Indeed denoising full-l…

@kumars , If you don't mind sending them to me that would be great! Just send them as a DM to me on here.

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denoise-ccs- loosing almost 60% of reads at denoising step of dada2

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