When annotatting the taxonomy of a 16S data, I met a problem. Could anyone help me? Thanks!
There is a 16S sequence data, which includes 3 pairs of primer amplified sequences. Firstly, I imported the data into qiime2, then I use q2-cutadapt to cut the primer and after joining paired end sequences and quality filter, I use deblur to obtain rep-seqs.qza and table.qza. until then, everything is ok ! But When I use q2-feature-classifier to annotate the taxonomy with [Greengenes 13_8 99% OTUs full-length sequences (https://data.qiime2.org/2019.7/common/gg-13-8-99-nb-classifier.qza), I found many sequences unassigned and many sequences only assigned to k__Bacteria, especially in several samples that are amplified using the same one pair of primer called a. Look at the picture below:
I want to know the reason why it happens, So I divided the 16S data into three parts according to the primer. After dealing with the 3 parts following the same work flow, I found the annotation of every part seemed right. Look at the picture below:
Then I used the function-merge-taxa to merge three parts. But it looks something false, there are still many sequences unassigned and many sequences assigned to k__Bacteria. Look at the picture:
It is difficult for me to solve the problem, I want to obtain the right annotation and get the differential taxa between two groups. Could anyone help me? Thanks!