Fungal ITS sequence from Illumina returns many unclassified reads

[linjie_0911 (Linjie Tang)]

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.

[SoilRotifer (Mike Robeson)]

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:

Fungal ITS classification with UNITE database

Hi @Hillary_Smith, Thanks for posting! And many thanks for checking out the other forum posts related to this issue first. I have a few questions to get started troubleshooting: What primers are you using? What is the length of the sequences that you are classifying? What types of samples are you analyzing? (just curious but it may be relevant here) One way to test your classifier would be to classify a subset (10 would be enough) of the same sequences that you used to train the classifier. …

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:

Fungal ITS analysis tutorial

Fungal ITS analysis, mock communities, and more fun NOTE: This tutorial was written in a QIIME 2 2018.11 environment. It is not guaranteed to work with earlier or later versions of QIIME 2. This tutorial was compiled as a working exercise for a QIIME 2 workshop in December 2018, and does not represent the only possible fungal ITS workflow with QIIME 2, or even a benchmarked protocol recommendation. See other tutorials, e.g., the q2-itsxpress tutorial for other fungal ITS analysis options in QI…

-Cheers!

[linjie_0911 (Linjie Tang)]

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!