Hello, I have shotgun sequences that, prior to using in QIIME, I mapped to the UNITE database so I could eliminate extra non-fungal sequences that I wasn't planning on using in the analysis. Then I ran through QIIME, but I'm getting a large number of unclassified fungal reads.
Here are the steps I've taken:
qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path manifest-pe.tsv
--output-path pe-demux.qza
--input-format PairedEndFastqManifestPhred33V2
qiime vsearch join-pairs --i-demultiplexed-seqs pe-demux.qza --o-joined-sequences demux-joined.qza
qiime vsearch dereplicate-sequences --i-sequences pe-demux.qza --o-dereplicated-table derep-table.qza --o-dereplicated-sequences derep-seq.qza
qiime vsearch cluster-features-de-novo --i-table derep-table.qza --i-sequences derep-seq.qza --p-perc-identity 0.97 --o-clustered-table table-clustered.qza --o-clustered-sequences rep-seqs-clustered.qza
qiime feature-classifier classify-sklearn --i-classifier ../../../../UNITE_database/sh_qiime_release_s_10.05.2021/developer/classifier.qza --i-reads derep-seq.qza --o-classification classify-nofilter.qza --p-confidence .97
Hello Rachel,
Welcome to the forums! 
Yes, something is up with that classification...
I appreciate the detail you provided about your methods so far. This part is especially strange to me:
After reads have been pre-filtered to match a database, it's pretty strange that they can't be classified against that exact same database!
I wonder if something is going wrong with that pre-filter mapping or maybe something is wrong with the sklearn classifier.
What program did you use to pre-filter?
How did you train the sklearn classifier?
Thanks for your response. I used Geneious to pre-filter. Here is what I did for training:
qiime tools import --type 'FeatureData[Sequence]' --input-path sh_refs_qiime_ver8_97_s_10.05.2021_dev.fasta --output-path UNITE_otus.qza
qiime tools import --type 'FeatureData[Taxonomy]' --input-format HeaderlessTSVTaxonomyFormat --input-path sh_taxonomy_qiime_ver8_97_s_10.05.2021_dev.txt --output-path ref-taxonomy.qza
qiime feature-classifier fit-classifier-naive-bayes --i-reference-reads UNITE_otus.qza --i-reference-taxonomy ref-taxonomy.qza --o-classifier classifier.qza
Thank you for telling me more.
Your classifier code looks okay. I've not used Geneious (Assembly and Mapping | Geneious Prime) for filtering but I imagine it's working as intended.
The unite database is targeted at the eukaryotic ITS region, so I wonder if your shotgun reads from full genomes are largely falling outside of this region.
When using Geneious to prefilter, did you use the exact same sh_refs_qiime_ver8_97_s_10.05.2021_dev.fasta as the target database or did you use something provided by Geneious? Different database files could cause this issue, which is why I ask.
Ah that could be it! I used the updated UNITE database for the pre-filter and last year's version in QIIME. I'll rerun with the updated database and hope that helps.
Using the correct UNITE fasta helped a bit, but still a significant portion is not identified past kingdom Fungi.
Hey, that's good progress!
After updating the pre-filter, what percentage of reads are filtered out because they do not hit UNITE?
Would you like to explore this more? While some plugins focus on amplicons from a specific region, other plugins like q2-shogun are designed for shotgun reads.
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