Ancom differential analysis

User Support
1

I do have 5 columns in metadata file: SampleID, Tissue (soil, root, shoot), Treatment (T1, T2, T3), Village and Region for all sample.

there are 3 tissue: soil, root and leaf.

I wanted to see differential abundance in all soil sample. I think I am making error in giving --P-where parameter, could you please suggest How should I correct it.

I used below command to filter and further process:

[qiime2_16s_analysis]$ qiime feature-table filter-samples --i-table table.qza --m-metadata-file metadata3.tsv --p-where "Tissue='Soil'" --o-filtered-table Soil-table.qza

Saved FeatureTable[Frequency] to: Soil-table.qza

qiime2_16s_analysis]$ qiime composition add-pseudocount --i-table Soil-table.qza --o-composition-table com-Soil-table.qza
Saved FeatureTable[Composition] to: com-Soil-table.qza

[qiime2_16s_analysis]$ qiime composition ancom --i-table com-Soil-table.qza --m-metadata-file metadata3.tsv --m-metadata-column Tissue --o-visualization ancom-Soil.qzv
Plugin error from composition:

All values the grouping are the same. This method cannot operate on a grouping vector with only a single group of samples(e.g., there are no 'between' variance because there is only a single group).

Debug info has been saved to /tmp/qiime2-q2cli-err-hvbdjtm7.log

Thanks

2

This error means you don't have multiple values in the Tissue column. This makes sense, since in an earlier step, you filtered your table to only include Tissue='Soil' samples. How can you compare tissue types when you only have one type? Options include filtering your table on some other column, or computing ancom on another column.

3

Hi @thermokarst,

I have given three different treatment to soil sample, so I wanted to see how T1, T2 and T3 treatment affect the microbial diversity in soil. Like this I want to repeat for root and leaf.

should I use OR to add it like this:

"Tissue='Soil' OR Treatment='Yes'"

Thanks

4

No.

If you want to see how the treatment impacted differential abundance across the samples in the filtered table, specify the Treatment column when running ancom, not the Tissue column.

So:

qiime composition ancom \
  --i-table com-Soil-table.qza \
  --m-metadata-file metadata3.tsv \
  --m-metadata-column Treatment \
  --o-visualization ancom-Soil.qzv
5

Thank you very much @thermokarst.
Now I understand it.

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6

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