Please check the two .qzv files. I have tried to use two classifiers to assign the rep-seqs from DADA2, but the ratio of “unassigned” sequences is still very high, which can not be assigned to kingdom.
PE300 was used to sequence the V4 region of all samples. The classifiers were trained using the Greengene database and the newest Silva database with my own primer sequences.
I have changed the parameters of DADA2 to retain longer reads with more bases, but it is not helpful to decrease the ratio of “Unassigned”.
It is worth noting that the “Unassigned” sequences only exist in the isotope-labelled samples.
I want to know how to deal with the these “Unassigned” sequences. For example, I want to do Lefse analysis to look for significant biomarkers, should I take these unassigned sequences into account?
Thanks in advance!