Hi, I am working with paired end reads that have been demultiplexed but have not undergone any other processing by the sequencing company (so primers/adapters are still attached). I imported using Phred33 manifest file. The graphs of the unjoined sequences are below:

Based on this comment, I ran the following DADA2 command, using a smaller truncation parameter for the reverse reads based on the graph **(Do I need to remove the primers/adapters first? I did not… I see from this post that you can set the truncation parameters to remove the primer sequences, but I’m not sure how to incorporate that into also trimming for quality/denoising)**:

$ qiime dada2 denoise-paired

–i-demultiplexed-seqs demux-paired-end.qza

–p-trim-left-f 0

–p-trunc-len-f 270

–p-trim-left-r 0

–p-trunc-len-r 120

–o-representative-sequences rep-seqs.qza

–p-n-threads 0

–o-table table-dada2.qza

The total frequency for my 53 samples is from the FeatureTable is: 4,467. Compared to the Moving Pictures Tutorial frequency of 157,298, I am not sure if my truncation parameters are too stringent (in fact for one of my samples, there is a frequency of 0)? Is 270 (forward) and 120 (reverse) reasonable based on the graphs above?

For paired-end analysis, is DADA2 or the recent paired-end tutorial better to use and for what reason?

Many thanks for the help provided thus far! Any guidance on the above from more experienced analysts would be very appreciated. Thank you!