Hello Qiime2 community.
I'm seeing a strange result when I look at 18S taxonomy. My samples are DNA extracted from a test pit dug 1.5 meters deep in East Iceland. Samples were taken every 10 cm. We're looking to see any evidence of human habitation - the test pit was dug right next to a known stone foundation, outside the house.
I'm running Qiime2021.4, and using the SILVA138 classifier downloaded from the Qiime2 website (I did not train my own - yet). I'm getting a very strange result when I look at Vertebrates:
Total # Reads: 928204
Reads classified as Bos Taurus: 144269 (a full 15% of the entire dataset)
Reads assigned to Vertebrates that are not Bos Taurus: 12
Has anyone else seen anything this strange? I'm guessing the problem is somewhere in the assignment of reads step. My next step will be to train my own classifier on my data, but I'm happy to get more advice on this.
For those interested, here is a table of the highest % reads:
% of total reads | Phylum | Class | Order | Family | Genus | Species | Species |
---|---|---|---|---|---|---|---|
15.54 | p__Vertebrata | c__Actinopterygii | o__Teleostei | f__Teleostei | g__Teleostei | s__Bos_taurus | s__Bos_taurus |
15.51 | p__Basidiomycota | c__Agaricomycetes | NA | NA | NA | NA | NA |
11.91 | NA | NA | NA | NA | NA | NA | NA |
7.48 | p__Peronosporomycetes | c__Peronosporomycetes | o__Peronosporomycetes | f__Peronosporomycetes | NA | NA | NA |
5.82 | p__Basidiomycota | c__Agaricomycetes | o__Agaricales | NA | NA | NA | NA |
5.79 | p__Phragmoplastophyta | c__Embryophyta | o__Magnoliophyta | f__Magnoliophyta | g__Magnoliophyta | NA | NA |
5.72 | p__Mucoromycota | c__Incertae_Sedis | o__Mortierellales | f__Mortierellaceae | g__Mortierella | NA | NA |
3.08 | p__Ascomycota | c__Leotiomycetes | o__Helotiales | NA | NA | NA | NA |
2.99 | p__Ascomycota | NA | NA | NA | NA | NA | NA |
2.40 | p__Cryptomycota | c__LKM11 | o__LKM11 | f__LKM11 | g__LKM11 | NA | NA |
2.20 | p__Cercozoa | c__Glissomonadida | o__Glissomonadida | f__Glissomonadida | g__uncultured | NA | NA |
1.94 | p__Ascomycota | c__Dothideomycetes | o__Pleosporales | NA | NA | NA | NA |
1.59 | p__Ascomycota | c__Archaeorhizomycetes | o__Archaeorhizomycetales | f__Archaeorhizomycetaceae | g__Archaeorhizomyces | s__Archaeorhizomyces_borealis | s__Archaeorhizomyces_borealis |
1.24 | p__Basidiomycota | c__Tremellomycetes | o__Filobasidiales | NA | NA | NA | NA |
1.09 | p__Cercozoa | NA | NA | NA | NA | NA | NA |
1.09 | p__Cercozoa | c__uncultured | o__uncultured | f__uncultured | g__uncultured | s__uncultured_Eimeriidae |
The code I ran:
qiime feature-classifier classify-sklearn
--p-n-jobs 8
--i-classifier silva-138-99-nb-classifier.qza
--i-reads Iceland-2019-STP-FWDs-rep-seqs-dada2-run3-18s.qza
--o-classification Iceland-2019-STP-taxonomy-silva-run3-18s.qza &
qiime metadata tabulate
--m-input-file Iceland-2019-STP-taxonomy-silva-run3-18s.qza
--m-input-file Iceland-2019-STP-FWDs-rep-seqs-dada2-run3-18s.qza
--o-visualization Iceland-2019-STP-taxonomy-silva-table-run3-18s.qzv
qiime taxa barplot
--i-table Iceland-2019-STP-FWDs-table-dada2-run3-18s.qza
--i-taxonomy Iceland-2019-STP-taxonomy-silva-run3-18s.qza
--m-metadata-file Skalanes-Test-Pit-Metadata.tsv
--o-visualization Iceland-2019-rSTP-taxonomy-silva-bar-plots-run3-18s.qzv