Dada2 analysis queries

[Nicholas_Bokulich]

Hi @Asha1,

@Asha1

Shouldn’t I follow Dada2 qiime work flow for ITS Sequence analysis?

Yes — we have a basic ITS tutorial that you can follow here (though note that as with 16S analysis there are many different possible workflows with QIIME 2 for analyzing ITS and you may want to check out plugins like q2-itsxpress and others to help with your ITS analysis):

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…

@Asha1

Would reduced sequence count affect further analysis like taxonomy identification?

Well maybe yes and maybe no. You need to check out the dada2 stats file to assess.

1. If you are losing these sequences at the filtering stage or any stage prior to read merging (joining) in the dada2 pipeline, then the loss should be random and will not impact taxonomic composition profiles or diversity estimates unless if loss is extreme.

2. If you are losing too many sequences at the merging step, then this loss will be non-random and will severely skew taxonomic profiles. ITS is hypervariable in length and sequences lost at the merging stage will bias for taxa with longer ITS that cannot merge. If this is the case, you either need to adjust your trimming settings or quite possibly only process your ITS data as single-end reads (e.g., only use the forward reads in your analysis).