Hi @llenzi
Thanks so much for your reply.
We sent our samples to a third party lab but they reported that sample quality was good and PCR cycles were 30-35 cycles
I followed your suggestion and I tried try the ‘–p-discard-untrimmed’ option.
qiime cutadapt trim-paired
--i-demultiplexed-sequences demux-paired-end.qza
--p-front-f CCTACGGGNGGCWGCAG
--p-front-r GACTACHVGGGTATCTAATCC
--p-discard-untrimmed
--o-trimmed-sequences trim-paired-demux.qza
--verbose
Now I was able to get all the sequences 280 bp long. So after that I tried different parameters but I was just able to improve my percentage of input non-chimeric (~31%).
qiime dada2 denoise-paired
--i-demultiplexed-seqs trim-paired-demux.qza
--p-trim-left-f 60
--p-trim-left-r 37
--p-trunc-len-f 278
--p-trunc-len-r 204
--p-max-ee-f 5
--p-max-ee-r 5
--o-table table-maxee.qza
--o-representative-sequences rep-seqs-maxee.qza
--o-denoising-stats denoising-stats-maxee.qza
--verbose
However, I have a few questions:
- After cutadapt step, my sequences are 280bp long, Does this mean I have to calculate a different trunc limit that the one I used before (truncating length should not go over 116 bp)?
- Do you have any suggestion about truncation and trimming parameters?
With the sample, I am not sure if it is the most representative sample, however I have a limited service units I can use to run my data so unfortunately I don't have other choice than try several times with only a few samples before running my whole data set.
Thanks so much,