Generating and analyzing mock communities

[Nicholas_Bokulich]

Hi @micro_guy ,

Great questions.

@micro_guy

I wanted to compare the community profiles for each region to the theoretical "ideal community" so that I could run 'qiime quality-control evaluate-composition'.

You are starting the in silico analysis at the wrong point:

@micro_guy

To do this, I downloaded the full-length 16S sequences from the product data sheet and generated an in-silico fastq file for the mock community

If you are trying to compare the observed vs. theoretical composition (as is the goal of evaluate-composition), then you should start with a feature table of the expected taxonomic composition (based on the taxonomic labels given in the chosen reference database that you are using for classification of your true observed sequences). To build this table you should take the list of species in your mock community (Zymo standard), find how these species are annotated in your reference database (SILVA 138), and create a table of their relative abundances in the mock community (example).

This tutorial is a bit old, but shows what this process should look like (though it does not show how the expected composition table should be constructed):

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…

This is a more direct way to get from point A to point B. Synthesizing a FASTQ, only to dereplicate etc adds a few unnecessary steps, which might also somehow distort the actual expected abundances.

Good luck!