I am seeking for help with solving a strange problem that I encountered for the first time. I used the following command to process my raw sequencing data, which were generated by using 16S primers 799F and 1193R (amplicon is about 400 bp).
qiime dada2 denoise-paired --i-demultiplexed-seqs Paired-end-demux.qza --p-trunc-len-f 280 --p-trunc-len-r 215 --p-trim-left-f 19 --p-trim-left-r 18 --o-representative-sequences rep-seqs.qza --o-table table.qza --o-denoising-stats stats-dada2.qza --p-n-threads 16 --verbose
Overall, i got good sequences after this step, and I also Blasted a few top sequences in NCBI and got bacterial hit/ with no problem. But when i used the following command to run the taxonomy step in Qiime2, the taxonomy file i got provided >90% unassigned results (txt file attached).
qiime feature-classifier classify-sklearn --i-classifier StinglessBee_16S_124/Analysis/silva-132-99-nb-classifier.qza --i-reads Pollination/Bacterial_analysis/seqs_01 --o-classification taxonomy_03.qza
taxonomy.tsv (1.6 MB)
I really don't know what was the problem causing this chaos, does anyone has any idea to solve it? Thank you in advance.