First Time Qiime-er needing guidance

[Hayley_Guay]

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-

wget
-O "pe-33.zip"
"filepath to zip"
wget
-O "pe-33-manifest"
"filepath to manifest, which I validated with Keemei"
unzip -q pe-33.zip

qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path pe-33-manifest
--output-path paired-end-demux.qza
--input-format PairedEndFastqManifestPhred33V2

qiime tools view paired-end-demux.qza

qiime demux summarize
--i-data paired-end-demux.qza
--o-visualization demux-pe-seqs-summary.qsv

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.

[colinbrislawn (Colin J Brislawn)]

Hello @Hayley_Guay,

I think you are off to a very good start! I can confirm that your imported data looks OK, and you should be ready to proceed with the next steps show in the various tutorials. On to dada2!

---

@Hayley_Guay

Any tips for choosing parameters for trimming/truncating based on my data?

Try this thread: Merging, Quality control and overlapping

---

@Hayley_Guay

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?

Try the original DADA2 docs! Pseudopooling is discussed in vivid detail on this page: Increasing the sensitivity of DADA2 with prior information

---

@Hayley_Guay

Any egregious errors in this process so far?

Not that I can see!

@Hayley_Guay

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.

I think you are doing exceptionally well, especially given that you are working with ITS data, which can be tricky. Let us know if you have any more questions along the way.