I have generated a Bray-Curtis PCoA that looks like a tripod with three columns that meet in the middle. I am curious if this indicates an error or what it means about the microbiomes I am analyzing. I am also curious if the bacterial taxonomic level can be set for the PCoA. To my understanding it is analyzing the bacteria at the species level of resolution.
Although I dont have a great answer for sure, my money would be that your rarefaction depth is too shallow. In that case, it looks like maybe you have a few dominant/abundance features that that's what's giving you the gradient along the axis. My rule of thumb is that I dont work with 16S data with fewer than 1000 sequences/sample and I prefer to go deeper. (2500-5000 is kind of my sweet spot between spending too much on sequencing and having enough data to analyze.) You might find a shannon rarefaction curve can help you in your decision: you want that line to plateau. Sometimes, that means samples get sacrificed.
The shannon rarefaction flattens out well. There are three samples out of the 210 that didn't want to level out very well but I don't want to lose a bunch of other samples because of those 3.
It looks like your data is just super shallow, since the rarefaction typically cuts around the median. I would recommend going back and checking a table summary and looking at your denoising stats.
This is not a depth that I would move forward with and seriously question as a reviewer.
Thank you for your response. I'm not sure what you mean by super shallow. My sequence counts vary a lot but before denoising I have a mean of 50,000 sequences. After denoising I have significantly less but still average 8,400 sequences per sample.
I read something in the Qiime forum that said different trimming lengths can create an issue but they only mention the left trimming. I have a left trim of 0 for all my reads. I only vary by a couple of bases on my right trim.
If you have a mean of 50,000 sequences/sample, then why are you rarefying at 508 sequences/sample? I mentioned my minimum rarefaction depth earlier... "shallow" has a relationship with that minimum depth
If you're not trimming your batches consistently, that could be another source of variation, although I would expect a slightly different confirmation for your PCoA in that case.
The sequence counts in my samples vary greatly. I already lose 21 samples when I rarify to 508. I tried rarifying to 1000 to see what would happen and I didn't gain much at all. Only 3 samples benefited from deeper rarefication. I figured it was better to retain samples.
Hi Justine,
I tried subsampling at 1000 and it didn't do much to the Bray-Curtis or Jaccard PCoAs. Maybe it really is just 3 major bacterial taxa driving the dissimilarities.
Thanks for testing that! If that's the case, you might expect it to be reflected either in a stacked barplot or maybe an empire plot. Those might both be good the check to see if it's a clade issue.
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