Differential abundance analysis (e.g. ANCOM) for paired samples (e.g. normal tissue vs. tumor tissue from cancer patients)

[Nicholas_Bokulich]

Neat study!

@sbslee

My current hypothesis for why ANCOM did not produce any significant hits earlier is because ANCOM did not use paired testing.

Seems plausible

@sbslee

Is it possible to perform ANCOM with paired testing?

Not at the moment

@sbslee

I’m getting apparently mixed signals (probably because some posts are older than others).

Yep! This is a matter of post age... in the first few releases of q2-composition it was technically possible to do a paired test with ANCOM but the test option was removed in later releases.

@sbslee

Are there alternative tools for differential abundance analysis that support paired testing?

Yes — not in QIIME 2 (as far as I know), but aldex2 would be a good solution in R, as a matter of fact here's the relevant documentation:

aldex.ttest: Calculate Wilcoxon Rank Sum test and Welch's t-test... in...

Perhaps @dgiguer can let us know if a paired test is also possible with q2-aldex2, but I could not find it from a quick search of the documentation.

Also not a QIIME 2 solution, but a very neat solution recently discussed by @jwdebelius and @mortonjt on this forum:

Songbird on paired samples

Hi all, especially @mortonjt , I’m trying to figure out Songbird on paired samples. Ive been through the tutorial and looked at the differential code from the original paper. Im slightly confused how to use this for paired samples, since as far as I can tell by the formula, the code is looking at a bulk comparison between before and after tooth brushing without conditioning on the individual. (The code is C(brushing_event) and I might have expected something closer to the (C(brushing_event) | h…

@sbslee

There are some other tools that got mentioned along my journey like DESeq2 and LEfSe. However, I wanted to ask the QIIME 2 community before digging in deeper on those tools.

I'm glad you did — see what @jwdebelius has to say about these:

Analysis workflow

Hi @Anas_S, Welcome to the forum! Just so you know, there's often a delay for the first couple of posts, so be patient, and it will appear soon. The place I would recommend starting is with one of the workflow tutorials. This presents a relatively complete analysis. My favorite is the parkinson's mouse tutorial, which will gety ou from raw fastq sequences to alpha diversity and a PCoA. If you have paired end sequences, you may want to look at denoising in either the Atacama s…

DESeq2 vs. ANCOM results

Hi @Parix, DESeq2 makes some assumptions about biomass load that let it side-step compositionality. Those assumptions may be true for RNASeq data, but have been shown to be less valid for microbiome data in recent years, which can lead to a high false positive rate. ANCOM is quite conservative. If you’re interested, you might check out some papers that have looked at this over the past few years: Weiss et al (2017) Normalization and microbial differential abundance strategies depend upon dat…