Hi. I want to denoise my paired-end-demultiplexed data with dada2.
I have trimmed the primers and adapters using cutadapt with your help :
qiime cutadapt trim-paired \
– i- *demultiplexed-sequences paired-end-demux.qza *
–p-front-f CCTACGGGNGGCWGCAG **
–p-front-r GACTACHVGGGTATCTAATCC **
I have read many of the questions in the forum and find out I have to keep about 12 bp and some normal variation.
according to PCR catalog: Order amplicon primers–The protocol includes the primer pair sequences for the V3 and V4 regions that create a single amplicon of approximately ~460 bp.
I think if I want to use both forward&reverse sequences I can use
–p-trim-left-f 0 **
–p-trim-left-r 0 **
–p-trunc-len-f 283 **
–p-trunc-len-r 247 **
(please let me know if I made a mistake in choosing them)
so I will have (283+247) - 460 = 70 overlapping region and its enough
I don’t know if there is a threshold for the percentage of input non chimeric sequences but I don’t think my results were enough as you can see in the picture:
also I tried these steps with dada2-single denoising in this way :
and I think it was better :
my question is that if I did all the steps correctly ( in using of cutadapt, identify trim & trunc parameters , etc.) which result is more reliable to continue? are the paired-denoise result enough or its better to use just the forward reads?
I’m really sorry for my long question but after I read many questions in the forum I couldn’t decide which one is better.
Thank you very much in advance for your time.