I understand working with ITS reads can be tricky, but I was looking for a second opinion on truncation. I have tried to denoise at many truncation lengths but I seem to only get 30-40% input in my denoising summary.
denoising-stats5.qzv (1.2 MB)
this is my most recent code that gave me the best results:
qiime cutadapt trim-paired \
--i-demultiplexed-sequences** /scratch/rylee650/demux-paired-end.qza \
--p-front-f ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGGTCATTTAGAGGAAGTAA \
--p-front-f ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTTGGTCATTTAGAGGAAGTAA \
--p-front-f ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCTTGGTCATTTAGAGGAAGTAA \
--p-front-f ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAACTTGGTCATTTAGAGGAAGTAA \
--p-front-r GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCGTTCTTCATCGATGC \
--p-front-r GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTGCGTTCTTCATCGATGC \
--p-front-r GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGCTGCGTTCTTCATCGATGC \
--p-front-r GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCATGCTGCGTTCTTCATCGATGC \
--p-adapter-f GCATCGATGAAGAACGCAGC \
--p-adapter-r TTACTTCCTCTAAATGACCAAG \
--p-discard-untrimmed \
--o-trimmed-sequences /scratch/rylee650/demux-trimmed.qza
qiime demux summarize \
--i-data /scratch/rylee650/demux-trimmed.qza \
--o-visualization /scratch/rylee650/demux-trimmed.qzv
qiime dada2 denoise-paired \
--i-demultiplexed-seqs /scratch/rylee650/demux-trimmed.qza \
--p-trim-left-f 0 \
--p-trim-left-r 0 \
--p-trunc-len-f 278 \
--p-trunc-len-r 225 \
--o-table /scratch/rylee650/table5.qza \
--o-representative-sequences /scratch/rylee650/rep-seqs5.qza \
--o-denoising-stats /scratch/rylee650/denoising-stats5.qza
qiime metadata tabulate \
--m-input-file /scratch/rylee650/denoising-stats5.qza \
--o-visualization /scratch/rylee650/denoising-stats5.qzv
my question then, is at what point am I truncating too much, and will it be possible to reach above 50% passing the input filter?
I have attached my demux trimmed and original demux file, feel free to take a look and see if everything looks proper in those files.
demux-trimmed.qzv (319.9 KB)
demux.qzv (313.6 KB)
TIA