ITS - fungal analysis

[andresbrun]

Hi all,
I'm conducting fungi community analysis for the first time. I'm basically following this tutorial.

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…

So far, I've only used the forward sequence of ITS1 primer (515F 5'-GTGCCAGCMGCCGCGGTAA)
I used DADA2 to denoise with these parameters:
--p-trim-left 10
--p-trunc-len 160
and the UNITE dB (unite-ver7-99-seqs-01.12.2017) for taxonomic identification

Of the total ~8,600 entries, only ~2,000 (less than 25%) were identified to a taxonomic level of order and below, whereas the remaining were only identified up to class or Phyllum (fungi) levels,
Is this expected? Is there any advice to improve the classification? for instance, during the denoise process

Many thanks,
Andrés

[colinbrislawn (Colin J Brislawn)]

Hello Andrés,

Fungi are less well studied than bacteria and ITS reads can vary in length, and this combination of reduced database coverage and variable length makes ITS classification perform worse than 16S classification at times. The short length of your reads specifically (150 bp total) would also reduce taxonomic resolution.

@andresbrun

Is there any advice to improve the classification? for instance, during the denoise process

Try using the new UNITE V8, which should be better than V7!

(I have some prebuilt UNITE v8.3 classifiers over here if that's helpful.)