• 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 (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 Mercator4 about)

  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

• 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 Mercator4 about)
  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 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
  • 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.

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).

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'