Fungal ITS sequence from Illumina returns many unclassified reads

Hi all,

I did a fungi community analysis with DNA extracted from food waste. It was sequenced with Illumina platform using ITS1&2.
Then I used Qiime 2 with SILVA132_99 gene database and UNITE 8.2 taxonomy database to classify them. This is what I got.

Judging by the possibility of finding a unclassified eukaryote, I BLAST all the entries from the sequence file.
And I got a quite different result.

I need some help with trouble shooting!

Thanks a lot guys.

1 Like

Hi @linjie_0911,

Not sure why you are using SILVA for ITS reads. SILVA only contains sequence data for SSU rRNA genes (i.e. 16S & 18S). So, I would not trust any classifications of ITS data resulting from a search against the SILVA db.

Could you provide more details on what primer sets you used? Did you merge paired-end reads, or just use the forward read? For example see this post of helpful questions:

Perhaps provide a list of commands, or simply the QZV file of the taxonomy barplot so we can look at the provenance information. Feel free to DM me if you do not want to share publicly.

Finally, if you search the forum you'll come across quite a few threads about Fungal ITS analysis. Here is one to get you started:



Hi @SoilRotifer,

Thank you so much for your thorough guidance!
This is the first time I touch hands on fungal taxonomy, I think I did get some fundamentals wrong.
I will try with the tutorial and with a new gene database!
Gotta love the support from Qiime community!

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