which result to choose in BLAST

[Nicholas_Bokulich]

Hi @Clarissa,
I moved your post to the "other bioinformatics tools" category, as it is fundamentally about interpreting NCBI BLAST results, so not a "user support" type question (for QIIME 2).

That said, it does touch on a user support issue with QIIME 2:

@Clarissa

A lot of uncultured or unassigned Organisms showed up in my Taxonomy Barplot file.

This is very often a technical issue, e.g., incorrect use of the classifier or problems with the reference database. See here for some troubleshooting steps:

Train you classifier - taxonomy

Hi @Jasmine84, See this post. Depending on the length of your sequences, the primers you are using, etc, deeper than order level might be unreliable. On 250 bp 16S rRNA gene sequences, we can usually get genus level and some species with a good degree of reliability. The naive bayes classifier in QIIME2 is designed to be cautious and reliable, to avoid false-positive errors, but you can lower the confidence parameter to make it less cautious. If you have: only kingdom-level classification, …

@Clarissa

So I wanted to manually determine the taxa using BLASTn. I chose to exclude Uncultured/environmental sample sequences and used “Highly similar sequences (megablast)”.

This is usually one of the first things I do for diagnosing this issue, too. Since you are getting lots of hits to Telonema I think it is worth asking: is this in your reference database? You can follow the troubleshooting steps I linked to above to determine if this is an issue relating to the query or reference sequences, or some other issue. Any time NCBI BLAST finds a good hit (as you have), q2-feature-classifier should in theory be able to as well and there is a user error keeping it from doing so.

@Clarissa

I am unsure of which result I should choose since most of them have the same Query cover. Is there even a right or wrong?

They all have the same query coverage, but a couple stand out as having much higher percent identity... and hence higher scores and lower E values than the others.

Good luck!