Help troubleshooting for defining best dada2 parameters

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Hi, the base quality of forward and reverse reads changed a lot after you trimmed the adaptors. Since the reads were already demultiplexed, I don't think they contain adaptor sequence. Maybe you can try trimming the primer sequence only and see if you get something different:

qiime cutadapt trim-paired\
  --i-demultiplexed-sequences paired-end-demux.qza\
  --p-cores 10\
  --p-front-f GTGCCAGCMGCCGCGGTAA\
  --p-front-r GGACTACHVGGGTWTCTAAT\
  --o-trimmed-sequences trimmed-paired-end-demux.qza

Alternatively, you can directly trim off the primer sequence using the --p-trim argument within dada2. Based on the quality score shown in demux.qzv, you can try running the following code and see what you get:

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs paired-end-demux.qza \
  --p-trim-left-f 19 \
  --p-trim-left-r 20 \
  --p-trunc-len-f 240 \ #  you can change to what you think is fit for the purpose
  --p-trunc-len-r 240 \ # you change to what you think is fit for the purpose
  --o-table table.qza \
  --o-representative-sequences rep-seqs.qza \
  --o-denoising-stats denoising-stats.qza

qiime metadata tabulate \
  --m-input-file denoising-stats.qza \
  --o-visualization denoising-stats.qzv

Check other posts in the forum (here and here) on primer trimming .

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