Dear community. I started working with Qiime2 (2023.2) recently and i am facing some issues with ANCOM/ANCOM-BC. I have read multiple posts in the forum and done the tutorials, but still not sure how to proceed. Originally, i was just running Alpha Diversity, Adonis and PERMANOVA test to analyze my samples (only the PERMANOVA test for the Treatments resulted significant (p=0.035)).
However i found this paper QIIME 2 Enables Comprehensive End-to-End Analysis of Diverse Microbiome Data and Comparative Studies with Publicly Available Data, where it was suggested to run additional analysis due to the nature of the sample (fecal samples from same animals on 3 different dates). Therefore i used it as a guide to continue my analysis.
When running ANCOM to determine which features differ in relative abundance, the volcano plot identified every feature as significant, even with low or 0 W. Here is my script
Filter features less than in 10% of Samples and 20 counts
!qiime feature-table filter-features
Add pseudocount to the filtered feature table
!qiime composition add-pseudocount
Run ANCOM to determine which features differ in relative abundance across the Treatments
!qiime composition ancom
I read here this problem could be work around using ANCOM 2 in R. I have no background in R. Then, i learned you could use ANCOM-BC on the last version of Qiime2 (2023.2 which i am currently using). At this moment is where i find new difficulties
(1) From this website, i tried to run ANCOM-BC on Qiime2, but failed as it says there is no ancom-bc command.
(2) Later i found in qiime the correct script. But when trying to run it with the filtered table with added pseudocount i get the following error message
!qiime composition ancombc
(1/1) Invalid value for '--i-table': Expected an artifact of at least type
** FeatureTable[Frequency]. An artifact of type FeatureTable[Composition] was**
(3) Here is where i have a few doubts, when running ANCOM-BC i dont need to filter and add pseudocounts, or filtering is still necessary?
Therefore i tried with just the filtered table from above and were succesful
!qiime composition ancombc
(4) However i cant find the script to generate a volcano plot from this artifact. I tried the next scrip as shown and got the following error message
!qiime metadata tabulate
There was an issue with viewing the artifact 'Run1/ANCOM/dataloaf.qza' as QIIME 2 Metadata:
** Artifacts with type FeatureData[DifferentialAbundance] cannot be viewed as QIIME 2 metadata.**
I found the script to tabulate the p-values and W. From what i see in the table, situation has not changed(recognizes all features as significant independently of W value).
!qiime composition tabulate
My biggest concerns are in point (3) and (4).
Hope i explained my situation clearly, i apologize if i used the wrong term, as i am still familiarizing myself with the pipeline.