# Interpreting ANCOM results
**URL:** https://forum.qiime2.org/t/interpreting-ancom-results/28985
**Category:** User Support
**Tags:** ancom
**Created:** [January 20, 2024, 5:28pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985 "2024-01-20T17:28:42Z")
**Posts on this page:** 6
**Page:** 1
### Author:  [@crw](https://forum.qiime2.org/u/crw)
#### Post date: [January 20, 2024, 5:28pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/1 "2024-01-20T17:28:42Z")
Hi QIIME 2 team!
I'm hoping you can help clarify some ANCOM results. I've searched the forums and think I understand that the W statistic is a count of the number of times the null hypothesis (groups are equal) is rejected and suggests which features are significantly different between groups. I think I also understand the percentiles based on the following post [Interpreting values from an ANCOM percentile abundance table - #10 by Lisa\_Crummett](https://forum.qiime2.org/t/interpreting-values-from-an-ancom-percentile-abundance-table/1497/10).
I have some confusion when interpreting a two group comparison where the ANCOM has pulled a feature out as significantly different but there seems to be overlap between the percentiles for the two groups. I've included two examples below. The first example is a 16S analysis that includes all the reads in the dataset (~200k reads). The second example is a subsampled version of the same dataset (~100k reads). In both examples ANCOM suggests the phylum Firmicutes is significantly different between the Normal and Tumor groups but I'm confused when I look at the percentiles to determine which group (Normal vs Tumor) has a higher abundance of Firmicutes. In the first example, the tumor group has the highest count of Firmicutes sequences (100%) at 78163 but the Normal group has a higher median (50%) at 39913. In the second example, the normal group has higher 100% and 50% abundances but the the 100% abundances are similar between the Normal and Tumor groups. Based on these results, which group is ANCOM suggesting has a higher abundance of Firmicutes in the first and second examples?
I'm running qiime2-amplicon-2023.9
Many thanks in advance for your assistance!
Caroline
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### Author:  [@gregcaporaso](https://forum.qiime2.org/u/gregcaporaso)
#### Post date: [January 22, 2024, 5:08pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/2 "2024-01-22T17:08:06Z")
Hi @crw,
In both cases, it appears that on average Firmicutes abundance is higher in the normal group, though it looks like you may have one or a few outliers in the tumor group that have abundances higher than any of the normal samples. If you were to sketch out a box and whisker plot where the top and bottom of the box were the 25 and 75th percentiles, and the top and bottom of the whiskers were the 0 and 100th percentiles, I think this would be clear.
Also, just FYI, we recommend using ANCOMBC now ([`qiime composition ancombc`](https://docs.qiime2.org/2023.9/plugins/available/composition/)), as opposed to ANCOM. There is a nice visualization for ancombc (`qiime composition da-barplot`) that can help with this interpretation.
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### Author:  [@crw](https://forum.qiime2.org/u/crw)
#### Post date: [January 22, 2024, 8:54pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/3 "2024-01-22T20:54:24Z")
Hi @gregcaporaso,
Thanks so much for your reply and the clarification! I'm working to update my analyses to use ANCOMBC but have a followup question. The QIIME 2 tutorials that included ANCOM recommended adding a pseudo count by creating a FeatureTable[Composition] artifact; however, ANCOMBC requires that "--i-table" be a FeatureTable[Frequency] artifact (the input for the FeatureTable[Composition] artifact). Do you recommend adding a pseudo count for ANCOMBC, or does ANCOMBC do this under-the-hood/ can tolerate frequencies of zero. I found one QIIME 2 forum post (link below) that suggests that a pseudo count may be added by ANCOMBC under-the-hood but any additional clarification is greatly appreciated.
Many thanks, Caroline
> [@Is a pseudocount necessary for q2\_ANCOM-BC?](https://forum.qiime2.org/t/is-a-pseudocount-necessary-for-q2-ancom-bc/22326):
>
> Please read the following before posting! Hello, Is it necessary to use a pseudocount when running data in q2-ancombc? I am not getting any error and results appear to be complete without pseudcount. Running with pseudocount gives similar results. However, when I generate differential plots in q2-QURRO, the sample plot is not being generated. It seems all taxa are represented in the feature plot, but I receive the following error for the sample plot: 0 / 20 samples (0.00%) currently shown. 20…
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### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [January 22, 2024, 9:40pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/4 "2024-01-22T21:40:05Z")
Hi @crw! 👋🏼
> [@crw](#):
>
> Do you recommend adding a pseudo count for ANCOMBC, or does ANCOMBC do this under-the-hood/ can tolerate frequencies of zero. I found one QIIME 2 forum post (link below) that suggests that a pseudo count may be added by ANCOMBC under-the-hood but any additional clarification is greatly appreciated.
Great find on that forum post! @colinbrislawn hit the nail on the head - a pseudocount is added internally from the R code that we use to wrap the ANCOM-BC method in q2-composition. The link he shared with supplementary discussion on why pseudocounts are needed is also a good resource to look over, if you haven't already!
Cheers 🦎
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### Author:  [@crw](https://forum.qiime2.org/u/crw)
#### Post date: [January 22, 2024, 10:09pm UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/5 "2024-01-22T22:09:56Z")
Great! Thanks so much for the quick reply and clarification, @lizgehret!
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### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [February 23, 2024, 4:10am UTC](https://forum.qiime2.org/t/interpreting-ancom-results/28985/6 "2024-02-23T04:10:23Z")
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