Contents of imported database

General Discussion
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Hello,

I'm trying to assign taxonomy to my sequencing data for the ITS region using RESCRIPt. When I manually use ncbiBLAST to assign my ASVs, I often pick up a genus that does not appear when I assign taxonomy using RESCRIPt using the NCBI or UNITE databases. Is there a way to search for a specific taxonomic assignment within a database?

Cheers,
Kat

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The problem with BLAST is that it will show all hits that are similar to your query. Often, the closest hits will not agree on the same specific taxa (e.g. hit different species. Often if the hits are equivalent, the results are arbitrarily sorted. So, the top items in the list are not necessarily the best hits. The classifiers will collapse the taxonomy to the lowest common ancestor if your query hits multiple reference sequences equally well. Especially, if the sequence is identical across different taxa.

Here are a few threads on this topic:

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Thank you for this response; it helps to understand how the function works. However, I'm unsure if the genus is even within the databases. It doesn't appear at all within my samples when they're highly likely to contain that genus. Is there a way to look at the taxa the database contains?

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@klunn, what genera are you looking for? All you need to do is take the taxonomy file (used to make construct the classifier) and view it like so:

qiime metadata tabulate \
    --m-input-file  taxonomy.qza \
    --o-visualization  ttaxonomy.qzv

Then you can search for your genera of interest within the taxonomy. That should let you know if they are contained within that database for classification.

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Ah, thank you! This makes sense, I don't know why I didn't think about searching the visual file.. I'm looking for Ascosphaera, It doesn't appear in either of the databases I've made.

I've been using my F/R primer sequences to set my classifiers. I wonder if I'm filtering out this genus by doing so?

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6

Well, the taxonomy file should be based on the full reference database. When making an amplicon-region specific classifier, you basically use the full database taxonomy file along with the subsetted extracted amplicon sequences. So, if you are unable to find the taxon in question it might not be present in the reference database... or be under a different taxon label.

For example.. a quick google search suggests that "Ascosphaera" used to be called "Pericystis". Does it exist under that or another label?

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Ohh okay, I initially checked my classifier file and that is where I can't find the genus. I checked the original database and I can see 13 entries of the Ascosphaera genus. I must be cutting out too much when I'm making my classifier. I'll look into other ways to trim the database!

Thank you for your help :slight_smile:

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