Abnormal high percentage of unclassified bacteria

[Abdelhady_Elshal]

Qiime2 version: 2023.9 installed by conda
database use for classification. GTDB

Beginner here.
I am getting around 50% of unclassified bacteria (p__Bacteria;;;), and I am assuming it's a problem because that's not usually the case. I used dada2 for denoising

$qiime dada2 denoise-single
--i-demultiplexed-seqs mergedseqs-goodfmt.qza
--p-trunc-len 280
--p-trim-left 40
--o-table dada2-table-280-40.qza
--o-representative-sequences dada2-seqs-280-40.qza
--o-denoising-stats dada2-stats-280-40.qza
--p-n-threads 8

$qiime feature-classifier classify-sklearn
--i-classifier SILVA515806_138_99_classifier.qza
--i-reads dada2-seqs-280-40.qza
--o-classification class-seqs-280-40.qza
--p-n-jobs 8

$qiime taxa filter-table
--i-table dada2-table-280-40.qza
--i-taxonomy class-seqs-280-40.qza
--p-exclude mitochondria,chloroplast,unassigned
--o-filtered-table filt-class-table-280-40.qza

Could you please help me see where the problem is?
I apologize in advance for the missing info. first time posting. Please feel free to ask for any!
Thank you!

[SoilRotifer (Mike Robeson)]

Hi @Abdelhady_Elshal, welcome to :qiime2: !

If you search the forum, you find that the most common reason for this issue, is that your reads are in mixed orientation. Here is at least one thread that should help get you started:

train a greengenes 2022 classifier for forward reads only

Hi @mawa86, If the majority of your reads are being assigned as "Unassigned" and "k__Bacteria", it could mean that your reads are in a mixed orientation, with respect to the classifier. That is, the both the forward and reverse reads appear in the R1 files, and similarly, in the R2 files. You can read many forum threads about this here. One way to sanity-check if this is the case, is to run qiime feature-classifier classify-consensus-vsearch ... as vsearch does not care about read orientatio…