Possible Analysis Pipeline for Ion Torrent 16S Metagenomics Kit Data in QIIME2?

[cjone228 (Carli Jones)]

Hi all,

I am working on the analysis for the ATCC mock communities using the above mentioned pipeline:

@cjone228

Possible Analysis Pipeline:

1. Import fastq files using manifest file format
2. Perform separate DADA2 step for each sequencing run, then merge DADA2 results

• Use denoise-pyro option
• p-trim-left 15
• Do not truncate`

3. Explore feature table
4. Create phylogenetic tree using SEPP (fragment insertion)
5. Alpha rarefaction and selection of rarefaction depth using diagnostic curves
6. Core metrics analysis (only metrics which include phylogeny)

• alpha diversity --> Faith’s PD only
• beta diversity --> Weighted and Unweighted Unifrac only

7. Taxonomic classification (from feature table in step 3) using pre-trained full length 16S Green Genes classifier

I completed steps 1-3, and am now working on the sepp fragment insertion step. In case anyone is curious, here is a picture of the quality plot we got in our demux.qzv file:

We then continued with the DADA2 step (using denoise-pyro) and got a feature table:

and rep-seqs file:

I am now encountering an error when trying to do the sepp-fragment-insertion step - we use MARCC, a cluster, in order to do a lot of our work, so the below screenshot is the output of a SLURM job that we submitted for this.

I realized that the code I was using from the sepp-fragment-insertion step from the Parkinson's Mouse tutorial is from QIIME2 2019.10, so in the 2019.7 version there is no option for --i-reference-database. I installed (or I think I installed) QIIME2 version 2019.10 into our MARCC account to fix this, but it still gave me the same error saying that this is not an option for an argument. Any ideas? Thank you so much!!

Carli