The compound set is a list of compounds you are interested in (e.g. a set of metabolites identified in your experiment). Either you can copy/paste a list of IDs, or upload a file (MBROLE expects tabs or new lines as ID separators).

See the Conversion section for further information on supported IDs.

Annotations are organized into several vocabularies extracted from metabolic and chemical databases. Select the vocabulary (or vocabularies) you want to use for the enrichment analysis. You can include multiple annotations at once by clicking on them while pressing Ctrl button. You can also use the box for searching annotations. Annotations already selected will appear in lighter color in the menu. To remove an annotation or vocabulary from the list press in the "x" besides it.

The vocabularies included in this version of MBROLE are the following:

MBROLE needs a background set of compounds to compute statistics. This background set is a reference used for assessing the significance of a given annotation to be enriched in your compounds of interest.

A given annotation could have a high frequency in your set of compounds simply because it is frequent in the whole metabolome. This is why a reference set is needed for assessing significance. This background set should contain all the compounds that could hypothetically be identified in your experiment (in a global profiling experiment, the background set will correspond to the whole metabolome).

Therefore, you can choose which type of background set is better depending on your data and/or the information you want to obtain:

In this new version, results are separated in two different categories. These categories are the same used to organize the vocabularies in the Annotations section of the analysis page. A new navigation bar allows you to change between the results obtained from (Direct and Indirect) annotations. Each of these sections includes a single table which can be sorted by clicking on its headers. By default, these tables are sorted by decreasing significance (increasing FDR). Each annotation (keyword) will be arranged in a row with several columns containing all the information:

1. Annotation shows the annotation (keyword) itself and, if available, a link to its entry in the source database.
2. Category shows to which vocabulary the keyword belongs (take a look at the classification of annotation vocabularies in the Annotations section of the user manual).
3. Set shows the number of compounds in your input that have annotations in that category.
4. In set shows the number of your compounds that have this particular annotation.
5. Background shows the total number of compounds with annotations in that category.
6. In background shows the number of compounds from the background with this particular annotation.
7. p-value of the enrichment analysis test for that annotation. It is calculated using the values from Set, In set, Background, and In background.
8. FDR shows the adjusted p-value calculated as False Discovery Rate (FDR).

The following two columns show up if you select "Show compounds in results table" at the top of the page.
9. Submitted IDs Shows those IDs from your set with that particular annotation (keyword).
10. Matching IDs Shows the equivalent IDs from your set that actually matched the particular annotation. For example, if you provided KEGG IDs and selected Pathways from HMDB as vocabulary, this column will show the equivalent IDs from HMDB involved in the corresponding pathways.

MBROLE computes the statistical significance of each annotation found using the background set. This is provided as a p-value, or the probability of obtaining such a number (or more) of those compounds with a particular annotation in case we take a random set of the same size from the background set.

This p-value is also adjusted for multiple testing using the false discovery rate (FDR) method by Benjamini and Hochberg (1995)^*. Both, p-value and adjusted p-value are reported for each annotation and, when possible, annotations and compound IDs are hyperlinked to their corresponding source database.

For KEGG pathways, the compounds in the input set are highlighted in the pathway representation generated by the hyperlink. P-values are colored based on their significance level, (green for values <0.001; red for values ≥0.05 and orange for the rest). Put the mouse over each "Category" cell and it'll show the number of metabolites used as background as well as the number of associated metabolites found in it. You can see an example of this in Fig.2 of the Tutorial

For practical purposes, MBROLE only shows those annotations found in three or more metabolites of the input set.

In case your query does not return any significant result and/or you are interested in those annotations filtered out, you can download the whole set of results clicking on the ("Download all annotations" button). The file generated is a TSV plain text file ready for being further processed with any spreadsheet program .

* Benjamini, Y. and Hochberg, Y. (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B. 57: 289-300.

Generation of plots

You can now choose among two different types of ID conversion when running an analysis:

• Direct conversion (option selected by default): searches for equivalent IDs when the source database of the annotation selected provides corresponding cross-references. When this option is selected, you will need to indicate the type of ID and the list of annotations will be updated disabling those whose source databases do not include such cross-reference.
• Automatic conversion: allows mixing IDs from different sources. If the source database of the annotations selected does not include cross-references of the input IDs, a subsequent search will look for an intermediate chemical ID that relates the input ID and the IDs used for the corresponding annotation.

MBROLE includes an independent conversion utility that implements the Direct Conversion described before. This utility also accepts chemical names as input for converting them to any of the supported IDs, and it is accessible via the "Conversion" link at the top of MBROLE pages.

Supported IDs

• BioCyc compounds (e.g. CPLX3O-77 for phosphofructokinase).
• CAS Registry Number (e.g. 26566-61-0 for galactose)
• ChEBI accessions (e.g. CHEBI:34590 for bromobutide).
• ChemSpider IDs (e.g. 733 for glycerin)
• ECMDB metabolites (e.g. ECMDB00039 for butyric acid).
• HMDB metabolites (e.g. HMDB0000064 for creatine).
• KEGG (e.g. C00008 for ADP).
• LIPID MAPS^® Structure Database IDs (e.g. LMST01010001 for cholesterol)
• MeSH chemicals (e.g. D005947 for glucose)
• PubChem Compounds (e.g. 5793 for glucose)
• TTD (e.g. D0J7XL for Gramicidin D).
• YMDB metabolites (e.g. YMDB00003 for urea).

MBROLE integrates information from multiple databases including chemical and biological information related to chemical compounds. In most cases these databases are public and their contents are freely available for non-commercial use. This is not the case with BioCyc and KEGG which require a paid subscription to access their most recent data.

For KEGG, data was licensed under an academic subscription for organizational use, and for BioCyc database collection, we used the versions available free of charge. Also, each database may be subject to specific terms of use, which can be found on their website.

This version of MBROLE analyzes annotations from the following databases:

• Kyoto Encyclopedia of Genes and Genomes (KEGG), release 102 (April, 2022)
• Human Metabolite Database (HMDB), version 5.0
• Yeast Metabolite Database (YMDB) (we used data from version 1.0 as we couldn't access a complete database file of version 2.0).
• PubChem, release August 2022.
• BioCyc database collection (Tier 1 and Tier 2), v.26 (EcoCyc and MetaCyc) and v.24 (rest of databases).
• Medical Subject Heading (MeSH), release from 2022.
• Chemical Entities of Biological Interest (ChEBI), release August, 2022.
• E.coli Metabolome Database (ECMDB), version 2.0.
• Therapeutic Target Database (TTD), update of September, 2021
• Comparative Toxicogenomics Database (CTD). Terms of use: [Link].
• PharmGKB, retrieved August 2022.
• PANTHER Knowledgebase, release 17.0.
• UniProt, release 2022_01.
• LIPID MAPS^® Structure Database (LMSD), release from November, 2022.
• PathBank, release from 2020.
• Reactome, version 82.

The dynamic interface of MBROLE is the result of combining PHP, JavaScript and CSS. Additionally, the following libraries are used: Chosen (version 1.1.0) for the selection of annotations and tablesorter (version 2.31.3) for sorting the table of results.

All data integrated in MBROLE is stored in a relational database that is queried based on the user input. The statistical calculations and the generation of plots are handled by Python scripts.

You can find more details in the publication of this version of MBROLE.

Browser Compatibility

The following document contains two detailed use cases to highlight the potential of the server in real-world analysis scenarios: mbrole3_use_case

We are using this set of 39 metabolites that includes KEGG compounds and MeSH descriptors.

Upload the file containing the metabolite IDs or copy and paste them in the text area. Note that IDs should be separated by tabs or new lines. Also, as there are chemical IDs from different sources, check the option: MBROLE automatic conversion.

In the next section, Annotations, choose the following annotations: Biological Roles (ChEBI), Diseases (KEGG), GO Terms (CTD), Pathways (KEGG), MeSH terms-MeSH chemicals and Panther Protein Class (HMDB).

In the section Background Set select Organism dependent. A drop-down list with all the organisms available appears. Search and select Homo Sapiens

Click on the Enrichment analysis button to start the analysis.

Once calculations have finished, you will get a results table with six columns summarizing results. The table is sorted by increasing "FDR" (from most to least significant). Each row contains information for a single annotation as it was explained in the Results section of the user manual. Results for Direct and Indirect annotations are separated in different sections.

1. Hovering the mouse over "p-value" and "FDR" columns reveals an image with the boundaries used to assign the color codes.
2. Put the mouse over each "Category" to show info about the background used to calculate the statistics.
3. Follow the link of the annotation to open its content in the source database.
4. Click on "Download result table" button to export all the results to a spreadsheet program.
5. Click on "Generate dot plot" or "Generate bar plot" to create plots with the most significant annotations of each category.
6. Click on "Indirect Annotations" to change to the results for the other category of vocabularies.

This set of metabolites is given in terms of chemical names so they cannot be used directly in the analysis. In this example we will convert them to HMDB, KEGG and ChEBI IDs so as to perform the analysis with them.

Go to the Conversion utility and upload the file or copy-paste its content and select: "ChEBI compounds", "HMDB metabolites" and "KEGG compounds". After you click the "Search IDs" button you will get a table with four columns (see Fig. 3). The table includes the submitted metabolite, the equivalent ID(s) and its source database.

This table can be downloaded as a TSV text file clicking on "Download results" button. Once checked that the name to ID conversion is OK (and eventually corrected), you can copy/paste the column with the IDs of the compounds into the Analysis form to analyze them.