Hi everyone,
I’m working on 16S rRNA amplicon data from potato stems using the 515F/806R primers. I’m using QIIME 2 version 2024.2.0. I’ve tried both paired-end merging and reverse-only reads (since the reverse reads have better quality). However, I’m getting no assignments (or just very high-level classifications) when using the SILVA database — and I see the same results if I directly submit sequences to the SILVA website. So it doesn’t seem to be a QIIME 2 classifier issue specifically.
What’s puzzling is that if I randomly pick some of these sequences and run them against NCBI BLAST, I get high-coverage, high-identity 16S rRNA hits to known bacterial species. Does anyone have insight into why SILVA would fail to classify these sequences while BLAST finds clear bacterial matches? And how can I improve my SILVA-based taxonomy assignments in QIIME 2 to better match what BLAST shows?
Thank you in advance for any suggestions!
rep-seqs.qzv (742.7 KB)
taxa-bar-plots.qzv (396.0 KB)
Hagai
1 Like
Hi @hraanan,
I wonder if your R1 and R2 reads are flipped / oriented incorrectly. There are other forum threads you can search related to this issue. Can you try running qiime rescript orient-seqs... command on your rep-seqs.qza file, then re-classify? Try with and without the --i-reference-sequences flag. If this is not provided then all sequences will be reverse complimented. Otherwise each read will be oriented based on how well it matches the provided reference alignment, i.e. SILVA*. These files are located here.
The issue is that the sklearn classifier requires that the sequence data match the orientation of the reference sequences. You can also use qiime feature-classifier classify-consensus-vsearch ... which does not care about orientation. However, if your reads are actually in mixed orientation... that is some are sequenced correctly (R1 is really the forward read, and R2 is really the reverse read), and others are not (R2 is really the forward read and R1 is really the reverse read), then any phylogenies, comparisons, etc, will be incorrect.
3 Likes
Thanks, I will try this.
Hagai