Mercator4

online tools

legacy versions

© 2018-2024 Usadel lab
IBG-4, Forschungszentrum Jülich
Heinrich Heine University Düsseldorf

  

Job submission for protein annotation

  • optionally, verify the validity of the FASTA-format of the sequences by using the online
  • upload a file with the protein or cDNA sequences in a valid FASTA-format (the annotation of protein sequences is faster, for cDNA sequences all six reading frames will be tested)
  • specify the type of sequence, protein Protein or cDNA sequence DNA
  • optionally, include other protein annotations (see )
    Include Swissprot annotations additional protein description by Blast sequence comparison to Swissprot database
    Include Prot-scriber annotations additional protein description by Prot-scriber tool
  • optionally, provide a job name and an email address to which the annotation results will be sent

Result of protein annotation

  • list with a simple statistics on how many of the protein sequences were successfully categorized

    Submitted seqs (S) total number of user-submitted sequences
    Classified seqs (C) protein sequences put into Mercator4 protein categories
    Annotated seqs (A) sum of classified seqs (C) and sequences assigned to the pseudo-category BIN-35.1 (see )
    Occupied BINs (O) occupied Mercator4 protein categories for which the user-submitted set contains matching sequences
    BINs available (B) total number of all true Mercator4 protein categories (no pseudo-categories)
  • bar chart summarising the protein assignments across the top-level context descriptions
    Each bar represents a top-level context description and the percentage of its protein categories occupied by at least one protein from the submitted protein sequences.

  • bar chart displaying the distribution of protein lengths based on the differences to category-specific reference lengths
    Each bar represents the number of proteins having a certain length difference to the reference length of the corresponding Mercator4 category.

  • download of protein annotation files for further processing on your local computer

    mercator4_result_data_fasta.zip FASTA-format that contains the protein annotations
    mercator4_result_data.zip specific tabular format that is required for the and the MapMan desktop application

Visualize result in tree viewer

The TreeViewer shows the protein categorization visualized as hierarchical tree with annotation context descriptions as branch nodes and protein categories as leaf nodes.

  • click on Result Tree Viewer
  • select a user job to display the results in the tree structure
  • optionally, add pre-evaluated protein annotations from one or more of the listed reference plant species
  • click on the button Show checked data on tree
Expanding the tree diagram at a location of interest displays the protein count per species per category. A mouse-over on a count tab pops up the individual names of the categorized proteins.
The color of the square in front of a protein description indicates the encoding genome of the corresponding protein
  • encoded by the nuclear genome
  • encoded by the plastidial genome in most or all plant clades
  • encoded by the mitochondrial genome in most or all plant clades

Visualize result in heatmap viewer

The HeatmapViewer displays the comparison of two protein sets with protein categories as spots colored according to the comparison outcome.

  • click on Result Heatmap Viewer
  • select a data set proteome A (user jobs will be displayed at the bottom of the drop down list)
  • select a second data set proteome B for comparison from the second drop down list
  • click on the button Show protein comparison on heatmap
A mouse-over on a spot pops up the description of the protein category and its context.
The color of a spot indicates whether the protein category is present in one or both protein sets and whether one of the protein sets has more or less proteins assigned to that protein category.
  • not present in either proteome (default state before selecting the protein sets)
  • present only in proteome A
  • present only in proteome B
    • present in both proteomes,
    • both proteomes contain the same number of paralogs
    • proteome B contains 1 paralog more than proteome A
    • proteome B contains 1 paralog less than proteome A
    • proteome B contains 2+ paralogs more than proteome A
    • proteome B contains 2+ paralogs less than proteome A
In addition, a green or blue background-color of a spot indicates a non-nuclear encoding of the corresponding protein
  • encoded by the plastidial genome in most or all plant clades
  • encoded by the mitochondrial genome in most or all plant clades
The Heatmap Viewer creates diagrams in Scalable Vector Graphics (SVG) format which conveniently can be downloaded with a browser that has an add-on for SVG export installed (for example the add-on SVG Export).

Online legacy protein annotation tool

Mercator version 3.6 is an older release of the context-based annotation approach based on a different annotation framework (see publication). The Mercator version 3.6 online tool is still available but any active maintenance has ended.

Publication

Lohse et al. (2014)
Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data
Plant Cell Environ. 2014 May, 37(5): 1250-1258.

Online legacy protein annotation tool

Although it is recommended to use the latest version of Mercator4, it is possible to submit sequences to legacy versions. Please notice that the available older Mercator4 versions do no longer support the online tools and .

Online enrichment analysis of protein categories

The online Mercator4 enrichment analysis identifies protein classes that are over- or under-represented within the full set of Mercator4 protein categories (BINs). The method uses statistical approaches to identify significantly enriched or depleted groups of protein categories.

  • upload the Mercator4 mapping result file that maps protein sequences to Mercator4 protein categories (BINs)
  • select the type of the Fisher's exact test
    • the one-sided exact test - specify also the over-representation or the under-representation analysis
    • the two-sided exact test to perform both the over- and under-representation analysis
  • enter a False Discovery Rate (FDR)-adjusted p-value to specify the tolerated ratio of the false positive results to the total positive results
  • enter a list of Genes of Interest
  • enter a list of Background Genes
The format of the lists requires one gene identifier per line. The gene identifiers have to be identical to the identifiers used in the Mercator4 mapping result file.
For example, in a typical RNA-Seq experiment, the Genes of Interest refer to differentially expressed genes, while the Background Genes refer to non-differentially expressed genes.

Result enrichment analysis

When an enrichment analysis finishes, a tabular output is generated and displayed. It shows the Mercator4 protein categories found to be enriched or depleted along with a description. Click on the Download CSV button to download the table.

Below the table, the same information is visualized as interactive BIN tree diagram with nodes colored according to an enrichment

  • yellow nodes indicate over- or under-representation (two-sided test)
  • green nodes indicate over-representation (one-sided test)
  • red nodes indicate under-representation (one-sided test)
A click on a node toggles between expansion and collapsing of the subtree. The enrichment analysis creates diagrams in Scalable Vector Graphics (SVG) format which conveniently can be downloaded with a browser that has an add-on for SVG export installed (for example the add-on SVG Export).

Online validation of FASTA-formatted sequences

The FASTA-format is a text-based format for representing protein or nucleotide sequences. The FASTA validator allows users to test the FASTA-format of a sequence file before submitting it to Mercator4. Each record in the FASTA-formatted file will be validated and all records not supported by Mercator4 will be listed. Optionally, the user can check the Create Mercator4-valid FASTA file checkbox and download a Mercator4-valid version of the file with all records containing errors removed.

Requirements for a FASTA-formatted file

General requirements for a FASTA-formatted file

  • each entry in the file starts with > followed by the name of the record
  • the record name must be unique within the file
  • the maximal sequence length allowed is 25000 characters
  • the file must not contain a mix of nucleotide and protein sequences

Single-letter codes for protein sequences supported in Mercator4

  • A,C,D,E,F,G,H,I,K,L,M,N,P,W,R,S,T,V,W,Y,U,O,B,J,Z
  • X for 'any'
  • * for 'stop'
  • - for 'gap'
Single-letter codes for nucleotide sequences supported in Mercator4
  • A,C,G,T,U
  • R,Y,S,W,K,M,B,D,H,V for certain ambiguous assignments
  • N for 'any'

Mercator4 - an online protein annotation tool

Mercator4 is an online tool to assign functional annotations to protein sequences of land plants (including flowering plants, ferns, horsetails, mosses, liverworts, and hornworts). Mercator4 can also annotate highly conserved proteins among the green algae groups of Archaeplastida. The results from user-submitted protein sequences can be visualized online and/or downloaded for further analysis.

The Mercator4 functional annotations are designed as a hierarchical framework ("Mapman4 framework") with each child node term being more specialised than its parent node term. The framework has 31 top-level categories (see figure above) which end with the protein categories at the leaf-level. Protein sequences are only assigned to leaf-level categories but the annotation is based on the full hierarchical path including all levels.

Protein pseudo-categories

A protein's context and category is depicted as a hierarchical number. The first number of the hierarchy refers to one of the 31 Mercator4 top-level categories (see list in top figure). Protein sequences which cannot be categorized by Mercator4, are assigned by default to the top-level protein pseudo-category BIN-35 "no Mercator4 annotation" (the pseudo-category BIN-35 was introduced in an old framework developed for the MapMan desktop application, Thimm et al. 2004).

For a standard plant proteome, approximately 55% to 60% of the predicted protein sequences can be categorized by Mercator4. An option to increase the protein annotation rate is the annotation tool ProtScriber v.0.1.3 (Eiteneuer and Hallab, unpublished, available on GitHub). Another option is the alignment tool Blast by which the Swiss-Prot protein annotation of a similar protein is selected (Swiss-Prot dataset of Viridiplantae proteins). For an average plant proteome, ProtScriber and Swiss-Prot annotations are available for more than 60% of all the plant protein sequences, but most protein descriptions are less specific than Mercator4 protein categories.

Mercator4 protein (pseudo-)category Mercator4
protein category
other protein
description
BIN-1 .. BIN-30 or BIN-50 yes yes or no
BIN-35.1 | no Mercator4 annotation.other annotation available no yes
BIN-35.2 | no Mercator4 annotation.no other annotation no no

Protein function annotation results

When a Mercator4 job finishes, an overview of the results is displayed as

  • list that gives a simple statistics on how many of the protein sequences were successfully categorized
  • bar chart that summarises the protein assignments across the top-level context descriptions
  • bar chart that displays the distribution of protein lengths based on the differences to category-specific reference lengths (each reference length has been evaluated from the median length of matching proteins from ~250 or more land plant species)

The Mercator4 protein annotation results can be downloaded for

  • any further processing on your local computer ('mercator4_result.zip' and 'mercator4_result_data_fasta.zip')
  • usage in the MapMan desktop application ('mercator4_result.zip')
  • usage in the online ('mercator4_result.zip')

The protein annotations can also be visualized by two interactive online tools

  • TreeViewer shows the protein categorization visualized as hierarchical tree with annotation context descriptions as branch nodes and protein categories as leaf nodes
  • HeatmapViewer displays the comparison of two protein sets with protein categories as spots colored according to the comparison outcome

Mercator4 updates

The hierarchical framework for Mercator4 is regularly updated and extended. For details about the version history see the . Although it is strongly recommended to use the latest version of Mercator4, it is also possible to submit sequences to legacy versions .

  • latest version of Mercator4 is release 6 (Oct 2023) with more than 6200 individual protein categories

Contact & publications

For any questions and suggestions, please feel free to contact us (plabipd@fz-juelich.de).

  • Bolger et al. (2021)
    MapMan visualization of RNA-Seq data using Mercator4 functional annotations.
    Methods Mol Biol. 2021, 2354: 195-212.
  • Schwacke et al. (2019)
    Mapman4: a refined protein classification and annotation framework applicable to multi-omics data analysis.
    Mol Plant. 2019 Jun 3, 12(6): 879-892.

legacy versions
  • Mercator4 v.5 (July 2022)

    • updated: annotation framework
      • BIN-01..BIN-28 + BIN-30 consists of 7544 nodes
        • 29 top-level context nodes
        • 1782 context nodes
        • 5733 protein categories
  • Mercator4 v.4 (October 2021)

    • added: HeatmapView online tool
    • added: new top-level context (BIN-28 "Plant reproduction")
    • updated: annotation framework
      • BIN-01..BIN-28 + BIN-30 consists of 6897 nodes
        • 29 top-level context nodes
        • 1667 context nodes
        • 5201 protein categories
  • Mercator4 v.3 (July 2020)

    • added: download of FASTA-formatted annotation data
    • added: new top-level context (BIN-30 "Clade-specific metabolism")
    • updated: annotation framework
      • BIN-01..BIN-27 + BIN-30 consists of 6420 nodes
        • 28 top-level context nodes
        • 1573 context nodes
        • 4819 protein categories
  • Mercator4 v.2 (July 2019)

    • updated: annotation framework
      • BIN-01..BIN-27 consists of 5934 nodes
        • 27 top-level context nodes
        • 1456 context nodes
        • 4450 protein categories
  • Mercator4 v.1 (May 2018)

    • annotation framework
      • BIN-01..BIN-27 consists of 5427 nodes
        • 27 top-level context nodes
        • 1304 context nodes
        • 4095 protein categories

Can I submit a FASTA file containing both DNA and protein sequences?

No, this will result in an error. FASTA files submitted to Mercator4 must be exclusively DNA or Protein sequences.

Very few of my sequences are assigned to functional BINS. Why?

You should verify that that you have selected the correct sequence type (DNA or Protein) before submitting the mercator job. If you submit DNA sequences, but specify the type of sequence as Protein, an error will be not be generated, but very few sequences are likely to be assigned to functional BINs. If you are sure that you have selected the correct sequence type, verify that you have submitted gene sequences (introns must be removed). Mercator4 is designed for land plant protein annotations: if you submit sequences from non-plant organisms, the classification and annotation rate will likely be low.

I get an error that my sequences are incompatible with Mercator4 - what can I do?

Mercator4 has been upgraded to accept a number of ambiguous protein sequences. However, there are still certain criteria which a sequence has to meet. To validate your sequence for Mercator4, you can run your FASTA file on the button which will give a detailed report including the possibility to generate a Mercator4-valid FASTA file with the offending records removed.

I get an Unknown Error (or Internal Error or Server Error) when running my job. What does this mean?

We try to handle every error scenario and provide a detailed description why the job failed. If you experience such an error, please send an email to plabipd@gmail.com with the 'JOB ID' (Starts with GFA-XXXXXXXX).

I ran my sequences on Mercator4 six months ago, but now the version has changed. Can I run my sequences against an older version?

Yes. We provide which will allow users to run against older versions of Mercator4.

My job has been queued for hours. Is it really running?

This cluster is capable of running many jobs in parallel, but can still be overpowered if many users submit jobs simultaneously. If your job has been queued for hours, submitting the same jobs again will not speed up the process. If your job has not completed after 4 hours, then you should contact us at plabipd@gmail.com providing us with the 'JOB ID' .

My browser crashed while running a job, and now I cannot access my job any more. What can I do?

As we do not require users to login to submit a job, the only way we have to track your job is using a 'browser session'. If your browser has crashed, then a new session is created and the link to your jobs is lost. However, if you entered a email address when you submitted the job, you will still be notified (along with a link to the results) when the job has finished. If you did not enter an email address, but have taken a note of the JOB ID, then you can email us at plabipd@gmail.com to get the results.