Hello Qiime team and fellow researchers! Please take this down if this sort of request is not allowed.
I am an independent researcher using this program for the first time and I want to make sure I am doing everything correctly. I've already received some help within this forum getting my data imported and help with working through tutorials. I am hoping that anyone reading could take a look at the data I have and the commands I have run so far and just help me decide if I am on the right track.
I am using qiime2-2021.8. The samples I am working with are amplified ITS regions from soil samples, with multiple fungal species expected in each of my ten samples.
These sequence files came from GeneWiz NGS, and this is how they looked before I put them in a zip drive and manifest file:
I input the zip file according to the Fastq Manifest Phred33V2 protocol with these commands-
"filepath to zip"
"filepath to manifest, which I validated with Keemei"
unzip -q pe-33.zip
qiime tools import
qiime tools view paired-end-demux.qza
qiime demux summarize
This is what my demux-pe-seqs.qsv file looks like -
My specific questions are:
Did I choose the correct input format/path for the file set?
Am I set to continue analysis with qiime dada2 denoise-paired?
Any tips for choosing parameters for trimming/truncating based on my data?
Looking ahead, should I use --p-pooling-method Independent or Pseudo? Any resources for the difference here that is more in-depth than the docstring on the QiimeDocs page?
Any egregious errors in this process so far?
Like I said, I am relatively inexperienced with this analysis, so I'd appreciate any suggestions or resources anyone can think to offer. My experiment set-up is a bit different than what the tutorials offer, which makes me want to be extra careful with each step to make sure my data is valid.