# Alpha/Beta diversity plots and reporting specific samples/groups that show significant differences
**URL:** https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231
**Category:** User Support
**Created:** [November 10, 2023, 3:58pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231 "2023-11-10T15:58:49Z")
**Posts on this page:** 9
**Page:** 1
### Author:  [@dfitzer1](https://forum.qiime2.org/u/dfitzer1)
#### Post date: [November 10, 2023, 3:58pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/1 "2023-11-10T15:58:49Z")
Hello
I'd just like some advice regards interpreting my alpha and beta diversity analysis outputs. I have 8 compost samples (3 replicates each) chosen at different timepoints.
I have ran the Alpha diversity analysis for Shannon's diversity, Faith's phylogenetic diversity and Pielou's evenness and all report no significant differences amongst samples.
[9\_evenness\_group\_significance.qzv](https://forum.qiime2.org/uploads/short-url/kZjYh3o4Pf6hV8N2lkaUaJk6u3d.qzv) (437.7 KB)
[9\_faith\_pd\_group\_significance.qzv](https://forum.qiime2.org/uploads/short-url/8Vd3BRt8D1sSvg8ISmrIh4KqIIx.qzv) (438.4 KB)
[9\_shannon\_group\_significance.qzv](https://forum.qiime2.org/uploads/short-url/xAFxo0u3HxolppiNluDRcBGt8bM.qzv) (437.7 KB)
Hypothetically speaking, if there was a significant difference reported, how could I determine which samples are responsible? Would it make sense to look at the reported pairwise comparisons and the associated p-values and corrected p-values (q)?
When I do my beta analysis (bray Curtis and unweighted unifrac) on the same set of samples they both report a significant p-value.
[9\_bray\_curtis\_significance\_PW.qzv](https://forum.qiime2.org/uploads/short-url/soAloO4WMCyJwjsjQusdShsTiLa.qzv) (564.3 KB)
[9\_unweighted\_unifranc\_group\_significance\_PW.qzv](https://forum.qiime2.org/uploads/short-url/u6B8k7QStTUX2BamscapZL3N4TF.qzv) (575.7 KB)
Similar to the above is it possible to determine which specific sample comparisons are significantly different using these metrics.
Thanks in advance
David
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### Author:  [@jphagen](https://forum.qiime2.org/u/jphagen)
#### Post date: [November 13, 2023, 4:02pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/2 "2023-11-13T16:02:14Z")
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### Author:  [@jphagen](https://forum.qiime2.org/u/jphagen)
#### Post date: [November 13, 2023, 5:49pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/3 "2023-11-13T17:49:26Z")
HI @dfitzer1
> [@dfitzer1](#):
>
> Hypothetically speaking, if there was a significant difference reported, how could I determine which samples are responsible?
Yes, looking at the pair-wise comparisons would give you the metrics to indicate samples responsible.
However, looking at your data the replicates may be problematic. For Kruskal-Wallis, and most of the statistical tests in Qiime2, one of the key assumptions is independence across samples. Replicates have inherent dependance. You will need to work around this by creating models that account for dependance.
[This forum](https://forum.qiime2.org/t/how-to-take-into-account-sample-replicates-in-beta-group-significance-testing/25208) post regarding replicates should be helpful to you in treating your replicates.
> [@dfitzer1](#):
>
> Similar to the above is it possible to determine which specific sample comparisons are significantly different using these metrics.
The pairwise comparisons also will be helpful to you in this case for specific sample comparisons.
I hope this answer is helpful to you! Let me know if you have any other questions as you work through your analysis.
-Hannah
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### Author:  [@lizgehret](https://forum.qiime2.org/u/lizgehret)
#### Post date: [November 13, 2023, 5:51pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/4 "2023-11-13T17:51:54Z")
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### Author:  [@dfitzer1](https://forum.qiime2.org/u/dfitzer1)
#### Post date: [November 13, 2023, 9:15pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/5 "2023-11-13T21:15:20Z")
Hello Hannah
Thanks for responding
> [@jphagen](#):
>
> However, looking at your data the replicates may be problematic. For Kruskal-Wallis, and most of the statistical tests in Qiime2, one of the key assumptions is independence across samples. Replicates have inherent dependance. You will need to work around this by creating models that account for dependance.
I am not sure what you mean by problematic. Do you mean they are not independent as they are in triplicate? Would you suggest the 3 replicates for each timepoint be combined? I thought I had read elsewhere that this was not advised.
> [@how to deal with technical replicates](https://forum.qiime2.org/t/how-to-deal-with-technical-replicates/19837/2):
>
> Hi, Ming! Currently I am analyzing a rumen microbiome as well wave. I am treating rumen fluid samples from different cows as biological replicates. I did not pool DNA in one sample, neither I merge them in my table. To perform any statistical analysis, or to get an overview of microbiome it is better to have replicates, rather than pool them together.
I could be misinterpreting this and your explanation.
Thanks
David
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### Author:  [@cherman2](https://forum.qiime2.org/u/cherman2)
#### Post date: [November 15, 2023, 11:05pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/8 "2023-11-15T23:05:50Z")
Hi @dfitzer1,
> [@dfitzer1](#):
>
> Do you mean they are not independent as they are in triplicate?
Replicates can sometimes make statistical comparisons hard because we do not want to violate assumptions of independence. It's just good to be aware of that dependency as you continue!
Can you clarify your definition of replicates? Are these Technical Replicates (repeated measures of a sample) or Biological replicates (multiple samples from the same environment or subject)?
🐢
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### Author:  [@dfitzer1](https://forum.qiime2.org/u/dfitzer1)
#### Post date: [November 16, 2023, 8:44pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/9 "2023-11-16T20:44:12Z")
Hi @cherman2
Thanks for the response. They are Biological replicates.
Best
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### Author:  [@cherman2](https://forum.qiime2.org/u/cherman2)
#### Post date: [November 16, 2023, 8:57pm UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/10 "2023-11-16T20:57:51Z")
Hi @dfitzer1,
There shouldn't be any issues. I would just keep this dependency in mind as you are running analyses!
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### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [December 18, 2023, 2:58am UTC](https://forum.qiime2.org/t/alpha-beta-diversity-plots-and-reporting-specific-samples-groups-that-show-significant-differences/28231/11 "2023-12-18T02:58:43Z")
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