LETHAL ERROR: Requested feature <PEPTIDOGLYCANSYN-PWY> was missing or not stratified


I have an issue with humann_barplot with --last-metadata option. When I use the option --last-metadata in humann_barplot, I have an error message as follows,

LETHAL ERROR: Requested feature was missing or not stratified

In the abundance table, I checked there is PEPTIDOGLYCANSYN-PWY. I am not sure what “not stratified” means in the error message.

Without the option --last-metadata, it is working without an error.

Would someone let me know what the problem is?


This script assumes you have potential metadata rows at the top of your table (e.g. sample location, sample time, etc.) followed by the HUMAnN rows. The --last-metadata flag tells the script where the metadata ends based on the name of the last metadata feature. I am not sure why using/not using this flag affects your error, but the error you’re seeing means that there are no species stratifications for the pathway you selected, i.e. it only have a community total abundance.

This script requires a feature that looks like…


(for example.) If you don’t have stratified rows for your feature of interest, i.e. rows with headers containing |, then the script can’t plot that feature.

Hi Eric,

Thanks for your response.

I think that I have stratification in the joined table.

The following is an example of PEPTIDOGLYCANSYN-PWY.

PEPTIDOGLYCANSYN-PWY: peptidoglycan biosynthesis I (meso-diaminopimelate containing)
PEPTIDOGLYCANSYN-PWY: peptidoglycan biosynthesis I (meso-diaminopimelate containing)|g__Absiella.s__Absiella_dolichum
PEPTIDOGLYCANSYN-PWY: peptidoglycan biosynthesis I (meso-diaminopimelate containing)|g__Acidaminococcus.s__Acidaminococcus_sp_CAG_542

PEPTIDOGLYCANSYN-PWY: peptidoglycan biosynthesis I unclassified

I am not sure why using --last-metadata flag made an error.

I will try to figure out what the problem is with comparing a file of hmp_pathabund.pcl from humann tutorial.


You only need to invoke --last-metadata if you have metadata rows at the top of your table that you want to ignore. Is that the case here?