# ANCOM abundance table: more abundant in which treatment?
**URL:** https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884
**Category:** User Support
**Created:** [July 30, 2019, 12:53pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884 "2019-07-30T12:53:16Z")
**Posts on this page:** 20
**Page:** 1
### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [July 30, 2019, 12:53pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/1 "2019-07-30T12:53:16Z")
Hi there,
I'm a little confused about one of my results from ANCOM:
- [ANCOM-D37-T3-vs-T4-collapsed-level-2.qzv](https://forum.qiime2.org/uploads/short-url/2wsKeeg0SLd0yeIBrdfKGXbahuX.qzv) (452.9 KB)
Specifically the abundances of Firmicutes. I can't work out if it's higher in T3 or T4. I drew a bar chart using the percentiles given in the hope that it would help but I'm still none the wiser (apologies for messiness - I'm not a fan of rulers 🤣).
From this, it doesn't look like there is any difference between the two treatments - but maybe I'm missing something in the way that ANCOM calculates this?
Thanks so much in advance for you help! 🙂
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### Author:  [@Mehrbod\_Estaki](https://forum.qiime2.org/u/Mehrbod_Estaki)
#### Post date: [July 30, 2019, 8:27pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/2 "2019-07-30T20:27:05Z")
Hi @xchromosome,
You are correct to have concerns about this. This isn't the first time we've seen ANCOM identify features significantly different with very low W values. See [this post](https://forum.qiime2.org/t/ancom-low-w-taxa-identified-as-significant-issues-workaround-ancom2-code-instructions/6040) for more details. However, I should also mention that ANCOM is much more powerful for larger datasets, meaning if you were to compare your data at the Genus level instead of the Phyla you would have much more meaning results. You are only comparing 7 Phyla which means the max W value you can get is 6, which doesn't allow for alot of certainty. This may be why you are seeing differences that may not actually be true.
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [July 31, 2019, 12:09am UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/3 "2019-07-31T00:09:56Z")
> [@Mehrbod\_Estaki](#):
>
> ANCOM is much more powerful for larger datasets
So @xchromosome, you could use a t-test for small groups like phyla, then 'dive deeper' using ANCOM for more specific taxonomies. 👍
Colin
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### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [July 31, 2019, 1:35pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/4 "2019-07-31T13:35:23Z")
Thank you to you and @colinbrislawn for the replies. I wasn't sure which taxonomic level to look at for my analyses, so I have done all of them at levels 2, 5, 6 and 7. I read the post you linked to - so I understand why ANCOM isn't really suitable for phylum level comparisons. What about level 5/family?
> [@colinbrislawn](#):
>
> So @xchromosome, you could use a t-test for small groups like phyla
Sorry, this is probably a silly question but how exactly would you recommend I do this? I was under the impression that t-tests couldn't be used for microbiome data!
Thank you! 😀
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 1, 2019, 1:10pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/5 "2019-08-01T13:10:46Z")
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [August 1, 2019, 5:47pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/6 "2019-08-01T17:47:40Z")
> [@xchromosome](#):
>
> Sorry, this is probably a silly question but how exactly would you recommend I do this?
In R: [youtube example](https://www.google.com/search?q=t.test+in+r&rlz=1C5CHFA_enUS856US856&oq=t.test+in+&aqs=chrome.0.0j69i57j0l4.2698j0j7&sourceid=chrome&ie=UTF-8#kpvalbx=_CCJDXc--D4300wLA27v4Dw23)
In excel: [youtube example](https://www.youtube.com/watch?v=JFq0Ad2PqxM)
> [@xchromosome](#):
>
> I was under the impression that t-tests couldn’t be used for microbiome data!
...um, stats tests don't care about where the data came from (microbiome or otherwise) but they do have assumptions that need to be met for the result to be valid. For example, the t-test assumes a normal distribution, and if your data distribution isn't normal (microbiome or not) then you got to pick a different test.
I'm not a card-carrying statistician, so I think I'll let the experts answer your stats questions!
Colin
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 1, 2019, 6:37pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/7 "2019-08-01T18:37:21Z")
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### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [August 4, 2019, 1:22pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/8 "2019-08-04T13:22:37Z")
Thank you for the replies.
> [@colinbrislawn](#):
>
> …um, stats tests don’t care about where the data came from (microbiome or otherwise)
Oh, I know! Sorry, maybe I didn't explain that right! What I meant was that I thought t-tests weren't really suitable for comparing relative abundances like you would generate from microbiome analysis, and that that was the advantage that ANCOM had over them. I've always struggled with stats though.
So, given that a W value of 2 (as in my result above) is invalid, can I ask what you think of this result, which is the same table and treatment comparison but at family level instead, with W scores of 30 - 38? [ANCOM-D37-T3-vs-T4-collapsed-level-5.qzv](https://forum.qiime2.org/uploads/short-url/mFS7SrJwgnrXAnYBMQwwvBr2gWE.qzv) (455.6 KB)
Thanks so much!
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 7, 2019, 2:56pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/9 "2019-08-07T14:56:04Z")
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [August 7, 2019, 3:21pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/10 "2019-08-07T15:21:27Z")
Hello again,
> [@xchromosome](#):
>
> t-tests weren’t really suitable for comparing relative abundances like you would generate from microbiome analysis, and that that was the advantage that ANCOM had over them.
Good point! Compositional data has its own issues that can violate expectations of some tests. So let's dive into these new ANCOM results.
Here's how Jamie describes the ANCOM volcano plot:
> [@How to interpret ANCOM results](https://forum.qiime2.org/t/how-to-interpret-ancom-results/1958/3):
>
> For the volcano plot, you are looking at the W statistic on the y-axis, and the F-score on the x-axis. So basically the x-axis is summarizing the effect size difference of the given species between your treatment groups, and the y-axis is the strength of the ANCOM test statistic.
>
> What you want to get out of this sort of plot are the ASVs with a high F-score and a high W-statistic – in other words points that are close to the the top right corner. These indicate that an ASV is suspected to be truly different across the groups.
You have no points your top right corner... meaning that nothing is super different between groups.
Have you tried this test using all your features without grouping them at a higher taxa level? Grouping can hide cool trends of specific microbes, so performing this test yet a third time using all feature might work well. 🤷♂️
Colin
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 7, 2019, 3:55pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/11 "2019-08-07T15:55:46Z")
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### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [August 10, 2019, 1:14pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/12 "2019-08-10T13:14:09Z")
Hi Colin,
Thanks for that. I've just had a read at that thread where Jamie explained the volcano plot and noticed that the example in question looked a bit different to mine. It seems that poster was comparing multiple groups, so there is an F-score on the x axis in that case:
So I understand that. But in my graph, there are only two groups. My x axis looks different:
In [this post here](https://forum.qiime2.org/t/negative-f-statistic-values-in-ancom-output-after-filtering-low-abundant-taxa/3211/3), Jamie said that for the example given with only 2 groups,
> "Considering the volcano plot, it looks like there isn’t an F-statistic being run. When there are only 2 categories, just the clr mean difference is calculated (which is essentially a log fold change). If you have negative log fold change, that is indicative of decrease (since log(x) \< 0 for 0 \< x \< 1), whereas a positive log fold change is indicative of increase (since log(x) \> 0 for x \> 1)."
(Sorry, can't find how to link the quote from that post!)
So from what I can gather, it seems like features appearing in the top left of the graph are just as significant/valid/important as those in the top right, in this case where there are only two groups? Is that correct?
Thanks again 🙂
Lindsay
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [August 10, 2019, 2:53pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/13 "2019-08-10T14:53:39Z")
Hello Lindsay,
I'm a little out of my depth as I'm not a statistician, but let's start with the CLR statistic.
I googled CLR and it turns up CLR® Calcium, Lime, & Rust Remover, which is not a stat test 🤦♂️
* * *
In our context, CLR stands for **center log ratio** , which works like this :
> [@ANCOM clr results confusion](https://forum.qiime2.org/t/ancom-clr-results-confusion/8794/3):
>
> clr coordinate do not necessarily imply increase / decrease (since it is not possible do this in the first place). Instead, these coordinates represent log-fold change relative to the _average_ microbe.
[This paper](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695134/) goes into more of the math, and explains why CLR is helpful for samples with different numbers of reads.
* * *
> [@xchromosome](#):
>
> So from what I can gather, it seems like features appearing in the top left of the graph are just as significant/valid/important as those in the top right, in this case where there are only two groups? Is that correct?
~~Yep! That sounds correct to me. These microbes are _decreasing_ between groups.~~
**EDIT: I don't have any idea what I'm talking about. I think a statistician needs to 'qiime-in' to tell you how best to report this.**
Colin
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### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [August 13, 2019, 4:26pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/15 "2019-08-13T16:26:08Z")
> [@colinbrislawn](#):
>
> I googled CLR and it turns up CLR® Calcium, Lime, & Rust Remover, which is not a stat test
Haha, I had a similar issue when trying to Google gneiss for the first time 😆
> [@colinbrislawn](#):
>
> [This paper](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695134/) goes into more of the math, and explains why CLR is helpful for samples with different numbers of reads.
Thanks - I will check that out. I'm struggling to get my head around the concept of an "average microbe" and how the increasing/decreasing relative to that links in with treatment groups. Maybe that paper will help it click!
> [@colinbrislawn](#):
>
> EDIT: I don’t have any idea what I’m talking about. I think a statistician needs to ‘qiime-in’ to tell you how best to report this.
Haha no problem! I really appreciate you taking the time to help. I will wait patiently for an expert to arrive. 🚑
Thanks!
Lindsay
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### Author:  [@mortonjt](https://forum.qiime2.org/u/mortonjt)
#### Post date: [August 21, 2019, 3:16pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/16 "2019-08-21T15:16:03Z")
Hi @xchromosome, part of the problem has to do with the nature of relative data -- it is not possible infer absolute differences from relative data. This means it isn't possible to infer increase / decrease from relative data. But it maybe possible to infer which microbes increase / decrease the most, and simulations show that ANCOM maybe able to do this. See our paper here: [Establishing microbial composition measurement standards with reference frames | Nature Communications](https://www.nature.com/articles/s41467-019-10656-5)
Are you seeing separation in beta diversity? If PERMANOVA doesn't give you significant results, maybe differential abundance won't give you the insights you want.
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### Author:  [@xchromosome](https://forum.qiime2.org/u/xchromosome)
#### Post date: [August 28, 2019, 12:24pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/17 "2019-08-28T12:24:22Z")
Hi Jamie,
Thanks for your input. Yes, I had highly significant differences in beta diversity, although both the PERMANOVA and PERMDISP tests showed significance (the PERMDISP less often and less strongly though). There were large differences in alpha diversity as well. I'm looking at antibiotic treatment vs control so I am expecting some taxa to be different between the groups. I've used gneiss as well but I found it less useful.
Thanks for the link to the paper. I've read it and tried to understand as much as I can from it, although I'm struggling a bit to be honest! Is the basic principal the concept of finding out the true abundance of one species (say x) and using that to estimate the true abundances of all the others based on their ratio to species x?
Can I just ask one more thing? One of the taxa that keeps popping up in all my different comparisons between various groups is "Azospirillum sp. 47\_25". It shows up when looking at genera and species level. Species fine, but why is it listed as a genus? Why not just Azospirillum?
Many thanks again 🙂
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 30, 2019, 6:58pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/18 "2019-08-30T18:58:55Z")
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### Author:  [@mortonjt](https://forum.qiime2.org/u/mortonjt)
#### Post date: [August 30, 2019, 7:20pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/19 "2019-08-30T19:20:35Z")
> [@xchromosome](#):
>
> Thanks for the link to the paper. I've read it and tried to understand as much as I can from it, although I'm struggling a bit to be honest! Is the basic principal the concept of finding out the true abundance of one species (say x) and using that to estimate the true abundances of all the others based on their ratio to species x?
Not quite, if you don't have the proper experimental setup (i.e. flow cytometry, ...), and only relative abundances from sequencing, then no, you cannot estimate the relative abundances. The point of the paper is that you can't infer absolute differences, but you can infer relative differences and rank the microbes according to how much they have changed across conditions.
> [@xchromosome](#):
>
> Can I just ask one more thing? One of the taxa that keeps popping up in all my different comparisons between various groups is "Azospirillum sp. 47\_25". It shows up when looking at genera and species level. Species fine, but why is it listed as a genus? Why not just Azospirillum?
>
> Many thanks again 🙂
That I'm not as sure of - maybe @Nicholas_Bokulich would have a better idea.
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [August 30, 2019, 7:26pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/20 "2019-08-30T19:26:21Z")
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### Author:  [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)
#### Post date: [August 30, 2019, 7:49pm UTC](https://forum.qiime2.org/t/ancom-abundance-table-more-abundant-in-which-treatment/10884/21 "2019-08-30T19:49:40Z")
> [@xchromosome](#):
>
> Can I just ask one more thing? One of the taxa that keeps popping up in all my different comparisons between various groups is “Azospirillum sp. 47\_25”. It shows up when looking at genera and species level. Species fine, but why is it listed as a genus? Why not just Azospirillum?
That must be a peculiarity with your database, not something QIIME 2 is doing. I agree — it should not be listed as a genus. You should check, and even possibly amend, your database to confirm that is where that genus name is slipping in.
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