Hi,
So I got the results from an ANCOM test but most of the differential features (according to the W statistics table) are actually on the left top corner of the Volcano plot instead of the right top corner. Does this mean that I cannot use the results of this test?
Cheers,
Pablo
Hi @Pablo_V,
No, that's a perfectly. valid result. It just means that the features are more enriched in whatever group is represented on the left side of the log ratio. (Unfortunately, these aren't well labeled). So, if I define "left" as controls, then you just interpret this as. ASVs which are more abundant in controls.
That said, depending on the size of your dataset, I might consider applying a filter, because you have a lot of potentially sparse features. You lose some features, but you gain power on the ones you retain
Best,
Justine
Hi @jwdebelius,
Thank you for your prompt reply.
Is there any way to know which side represent what treatment of my dataset?
Also, I have filtered my data to a minimum frequency of 1,500 and 10 features per sample, according to what is mentioned here Filtering data — QIIME 2 2018.11.0 documentation
Should I do a more stringent filtering? And if so, how?
Cheers,
Pablo
Hi @Pablo_V,
I would check your interquartile ranges and see how the data is ordered. Unfortunately, I dont think there's an easy way to specify directionality.
I would check prevalence... I like a feature represented in 10% of my samples represented, so it does depend on your sample size.
Best,
Justine
Hi @jwdebelius,
I have filtered my data using phyloseq and then imported the biom table to run ANCOM. When I run the following command:
qiime composition ancom --i-table table-filtered-ancom-abund-comp.qza --m-metadata-file metadata_rockwool.tsv --m-metadata-column Diagnostic --o-visualization ancom-filtered-diagnostic.qzv --verbose
I get the following error:
Traceback (most recent call last):
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/q2cli/commands.py", line 328, in call
results = action(**arguments)
File "</Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/decorator.py:decorator-gen-480>", line 2, in ancom
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 245, in bound_callable
output_types, provenance)
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/qiime2/sdk/action.py", line 452, in callable_executor
ret_val = self._callable(output_dir=temp_dir, **view_args)
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/q2_composition/_ancom.py", line 46, in ancom
metadata = metadata.filter_ids(table.index)
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/qiime2/metadata/metadata.py", line 1105, in filter_ids
ids_to_keep)
File "/Users/pablo/opt/anaconda2/envs/qiime2-2020.2/lib/python3.6/site-packages/qiime2/metadata/metadata.py", line 198, in _filter_ids_helper
% (', '.join(repr(e) for e in sorted(missing_ids))))
ValueError: The following IDs are not present in the metadata: '021f9f6d5cea1fb4903d7e3b4db61d33', '0302ece4031dfaf3eac34ddbfc2c7d6f', '04b8b02dd86522f055095c2ef17286ad', '04ecfad5772d2e09a84a0f5ef460536c', '0551bb63d550ae8c8f89dacebda76822', '084f5bc37bfe2e673033d1d4c47d6377', '0a4c8bffc9aac53bd22ac6dd2c468c44', '0ab25b517ef52f4bf414661f7a6d5e9b', '0c579d21280801f02a641e1608606927', '0ddcd311e02f742e2e0e61ce02cf9c29', '10f7d3043212747fec7e8064ab2cd213', '1290e271fbc50c66e2cd8e437d0d4a44', '133862bc5656b433803da453daeb9375', '13e333f11c5a6224f6fea469b509306f', '14ff660452637cf706f1a97b7c9d2c57', '16a7d8ffc6f02f49aeadebb5f9a85829', '18a980ed927822f60eebf93600ee8ff4', '19573493f06e15f074f664ad7505ac52', '1c2b8892a0d7f828d72808cd01cfd199', '1de4896460562cae3be7c9d14bfe84cf', '1e104b993416995498346089f57fd3bf', '1e542d2112237ddb072880742ade6180', '20b3df08cd5fac2be26a2928155bab69', '220795da096a5acfebca378d68cfb00a', '23dd3ddcb8dfc1dc043713b988712ed0', '25c9884cd991eb1695f6a20546b4a231', 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Plugin error from composition:
The following IDs are not present in the metadata: '021f9f6d5cea1fb4903d7e3b4db61d33', '0302ece4031dfaf3eac34ddbfc2c7d6f', '04b8b02dd86522f055095c2ef17286ad', '04ecfad5772d2e09a84a0f5ef460536c', '0551bb63d550ae8c8f89dacebda76822', '084f5bc37bfe2e673033d1d4c47d6377', '0a4c8bffc9aac53bd22ac6dd2c468c44', '0ab25b517ef52f4bf414661f7a6d5e9b', '0c579d21280801f02a641e1608606927', '0ddcd311e02f742e2e0e61ce02cf9c29', '10f7d3043212747fec7e8064ab2cd213', '1290e271fbc50c66e2cd8e437d0d4a44', '133862bc5656b433803da453daeb9375', '13e333f11c5a6224f6fea469b509306f', '14ff660452637cf706f1a97b7c9d2c57', '16a7d8ffc6f02f49aeadebb5f9a85829', '18a980ed927822f60eebf93600ee8ff4', '19573493f06e15f074f664ad7505ac52', '1c2b8892a0d7f828d72808cd01cfd199', '1de4896460562cae3be7c9d14bfe84cf', '1e104b993416995498346089f57fd3bf', '1e542d2112237ddb072880742ade6180', '20b3df08cd5fac2be26a2928155bab69', '220795da096a5acfebca378d68cfb00a', '23dd3ddcb8dfc1dc043713b988712ed0', '25c9884cd991eb1695f6a20546b4a231', 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'f6d4f12f5952bf76014386202c1f6574', 'f81f78a345c6fbbdce40078aa2e1d44c', 'f94b7bf8ba0c95ada4451d11dd11f702', 'faf373c2d5dcceb7eca0a6492bd13105', 'fc6622c636a5210293fb2873fc4761d9', 'fccd274e718e26b32c71f188d97d9e59', 'fe067c1146518eacd1c5537f5232ed30', 'ffd660f96ba6668b2d8d86fc4e150862'
See above for debug info.
Most probably the error has something to do with the filtering but I am not sure how to fix it. Could you please help me out here?
Thank you!
Cheers,
Pablo
Hi @Pablo_V,
The error says that there are IDs in your table that it can't find in the metadata. My suspecion is that there was something wrong with your import, because they look like hashed sequece IDs rather than sample names. (Unless you're using hashed sample names, in which case, good for you!) So, I would look at the feature table that comes out of phyloseq and see if the sample names are rows or columns. If they're rows, you need to transpose the table before loading it into qiime.
Best,
Justine
Hi @jwdebelius,
Thanks a lot for your advice. I checked the feature table that comes out from phyloseq and indeed I needed to transpose the table.
Cheers,
Pablo
Hi @jwdebelius,
One more question. Is there a reason why I only got one side of the Volcano plot and not two sides like the examples from the Parkinson mice tutorial? In this case, it only gives me differentially abundant features for one of the two treatments. How could I get the same for the other treatment?
Cheers,
Pablo
Hi @Pablo_V,
Do you have one or multiple groups? If you have 2 groups, I think ANCOM only gives a one sided plot. The other option is that what you have is lower in your reference group and there is nothing else, but that seems unlikely.
Best,
Justine
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