I’m processing MiSeq libraries (2x300bp) on V3-V4 16S region with DADA2 and I tried different truncation length (–p-trunc-len-f and --p-trunc-len-r) and I was surprised by results. I first tried to truncate at 300 but algorithm returned an error. Then, I tried reads truncation at 0 (no truncation), 285, and 260 obtaining an increasing number of features (132, 172 and 462 respectively).
My questions are:
- Why does no sequence truncation (truncation=0) leads to fewer features than truncation at 285 bp? I would expect that features number would be inversely proportional to the truncation length.
- What analysis I can trust? I tried taxonomical classification of reads truncated at 0 and 260 using naive-Bayesian algorithm and SILVA database, and number of assigned features (excluding Unassigned) was really different (102 vs 320 features with most of the 102 features also detected in the second analysis).
Other DADA2 parameters were set as following:
qiime dada2 denoise-paired
Thanks for the support!