# How to compare the differences in the intestinal microflora of the two groups
**URL:** https://forum.qiime2.org/t/how-to-compare-the-differences-in-the-intestinal-microflora-of-the-two-groups/20526
**Category:** General Discussion
**Created:** [August 23, 2021, 12:53am UTC](https://forum.qiime2.org/t/how-to-compare-the-differences-in-the-intestinal-microflora-of-the-two-groups/20526 "2021-08-23T00:53:23Z")
**Posts on this page:** 2
**Page:** 1
### Author:  [@yokmok](https://forum.qiime2.org/u/yokmok)
#### Post date: [August 23, 2021, 12:53am UTC](https://forum.qiime2.org/t/how-to-compare-the-differences-in-the-intestinal-microflora-of-the-two-groups/20526/1 "2021-08-23T00:53:23Z")
Hello everyone.
We would like to analyze the stools of a certain population before and after the treatment to find out the bacteria that were changed by the treatment.
However, one of the problems with this is that if one person's intestinal bacteria changes dramatically, it may have an effect on the entire population.
(For example, if one person's Lactobacillus increases due to treatment, it may appear that the overall Lactobacillus is increasing even if it is not increasing in other people.
The larger the sample size, the less likely this problem is to occur, but since our sample size is small, we are trying to eliminate it as much as possible.
By using lefse, it seems to have the above problem, but in that case, is it ANCOM?
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### Author:  [@jwdebelius](https://forum.qiime2.org/u/jwdebelius)
#### Post date: [August 24, 2021, 5:17am UTC](https://forum.qiime2.org/t/how-to-compare-the-differences-in-the-intestinal-microflora-of-the-two-groups/20526/2 "2021-08-24T05:17:12Z")
Hi @yokmok,
I think you've got two issues you need to contend with here. One of your major challenges is going to be each person's individual microbial identity. The microbes you start with will influence the microbes you have to respond to treatment and the way they respond. (This is a whole arm of exploration we don't talk as much about.) I think most of your tests are going to be sensitive to sparsity, if not outliers. You might choses to handle this by filtering (i.e. retaining features that present in at least X samples). This will also improve your FDR. I think the log ratio transform may also help. Of course, these methods are also more conservative than LefSe, so they may struggle with a smaller sample size. I'll link to a post talking about some of these methods below.
Best,
Justine
> [@Differential abundance analysis (e.g. ANCOM) for paired samples (e.g. normal tissue vs. tumor tissue from cancer patients)](https://forum.qiime2.org/t/differential-abundance-analysis-e-g-ancom-for-paired-samples-e-g-normal-tissue-vs-tumor-tissue-from-cancer-patients/17649/2):
>
> Neat study! Seems plausible Not at the moment 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. 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: [https://rdrr.io/bioc/ALDEx2/man/aldex.ttest.html](https://rdrr.io/bioc/ALDEx2/man/aldex.ttest.html) Perhaps @dgiguer can let us know if a paired test is also possible…