• optionally, verify the validity of the FASTA-format of the sequences by using the online FASTA validation
• upload a file with the protein or cDNA sequences in a valid FASTA-format (for cDNA sequences all six reading frames will be tested)
• specify the type of sequence, protein or cDNA sequence
• optionally, add an additional protein annotation from the results of a Blast sequence comparison to the Swiss-Prot database
• optionally, provide a job name and an email address to which the annotation results will be sent

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

  Submitted sequences	total number of user-submitted sequences
  Classified sequences (C)	protein sequences put into Mercator4 protein categories
  Annotated sequences (A)	sum of (C) and the sequences for which a Swiss-Prot annotation is available (only relevant if the Swiss-Prot annotation option has been selected)
  Occupied Mercator4 categories (O)	occupied Mercator4 protein categories for which the user-submitted set contains matching sequences
  Mercator4 categories available	total number of the currently available Mercator4 protein categories
  Expected Length sequences (EL)	sequences whose length corresponds to the category-specific reference length
  Short Length sequences (SL)	sequences whose length is shorter than the category-specific reference length
  Long Length sequences (LL)	sequences whose length is longer than the category-specific reference length
  Summary	summary of the results in a nutshell (data in per cent)

• 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 Mercator4 enrichment analysis and the MapMan desktop application

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

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).

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
  • one-sided exact test - specify also the over-representation (enrichment) or the under-representation analysis (depletion)
  • two-sided exact test to perform both the over- and under-representation analysis simultaneously
• set the significance threshold by entering the False Discovery Rate (FDR)-adjusted p-value (e.g. 0.05) to specify the tolerated ratio of the false positive results to the total positive results
• define the input lists
  • Genes of Interest enter the list of identifiers you want to analyze (e.g., your Differentially Expressed Genes / DEGs)
  • example RNA-Seq experiment: list of differentially expressed genes
  • Background Genes enter the list of identifiers which represent the entire possibility space of your experiment
    • include ALL genes that were detectable/measurable in your experiment (e.g., all genes remaining after filtering for low counts)
    • ensure that the "Genes of Interest" are also included in the "Background Genes" list
    • avoid to use only the non-differentially expressed genes
    • don't use the entire genome if many genes were not expressed/detectable in the tissue examined or under the given conditions
    • example RNA-Seq experiment: list of all genes that had sufficient read counts to be tested for differential expression (detected genes), not just the non-significant ones
  • input format requirements
    • one gene identifier per line
    • gene identifiers have to be identical to the identifiers used in the Mercator4 mapping result file

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.

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'