For the first time user of SyMAP, read the System Guide to try the demo and learn details of the input files. This document provide details of parameters and functions.

Contents:

Project Manager

1. Build database

The projects selected from the Projects label in the left panel are listed in the Selected section on the right. The possible functions vary with the state, as listed below:

This section shows a table with the status of alignments between the selected loaded projects. Each cell in the table represents a pair of projects and the cell contains a status code showing whether or not that pair has been aligned (codes are listed below). Note that the table shows each pair cell twice, but only the lower cells are activated.

Clicking on a cell selects that pair of projects (the cell will be highlighted in green). Alignment&Synteny may then be computed or viewed for the selected pair using the function buttons.

Concat: By default, all sequences of the 1st genome are concatenated into one file and then multiple files are created from the 2nd genome to be searched against the first; the multiple files may have two or more sequential chromosomes concatenated together to create files that are >60M. To reduce memory usage, you can uncheck Concat so that multiple files are created for each genomes, and all files from the 1st genome are searched against the 2nd genome. See timing difference.

For the remaining display buttons, see User Guide.

2.a Overview

Click the Parameters link to open the Parameters window shown on the lower right.   Make sure the following parameters are set correctly before running the Alignment&Synteny (A&S) step. Load project: Group prefix, Minimum length, Sequence files Alignment&Synteny: Mask non-genes. If any of these need to be changed after A&S, then the alignment needs to be run again (note, the alignment can take a long time!!).   Sequence and Anno (annotation) files: If the files are put in the default locations, nothing needs to be entered for them. Default locations: data/seq/<project-name>/sequence data/seq/<project-name>/annotation

When a Group prefix is entered, it allows SyMAP to remove the prefix from the chromosome names and use the remaining part as a shorter name (e.g. '1' instead of 'chr1', as shown on the lower right).   If the sequence file has a mix of prefixes (e.g. Chr01, Scaf2345): If a Group prefix is entered, only sequences with that prefix will be loaded. Leave the Group prefix blank will load all sequences; their prefix will not be removed (e.g. the 'chr3' on the upper right).   Long names really clutter the display; it is best to use a consistent prefix that SyMAP can remove, otherwise, use really short prefixes, e.g. 's' for scaffold, 'c' for chromosome.

• You may remove the prefix after the project is loaded. For example, if your sequences had names "Scaf1", "Scaf2", etc, and "Scaf" was NOT entered as the Group prefix before load, you may enter it later and it will be removed from all sequence names. However, this is not reversible and a prefix cannot be added to the sequence names.

Minimum length
Minimum length of a group sequence to load; smaller sequences will be ignored. Note that annotations for ignored sequences will also be ignored, but some error messages will be printed to the terminal.

Sequence files
Select the input FASTA sequence file(s), or directories of sequence files. For formatting, see Sequence files. Default location: data/seq/<project-name>/sequence

If any of the above 3 are changed:
▸ If the project has already been loaded, Reload Project.
▸ If A&S has previously been run, select Clear pair and remove the alignment files, then run A&S.

Anno files
Select the input GFF3-formatted annotation files corresponding to your sequences. Note, using a GFF3 file directly can cause problems; see Annotation files. Annotation is optional but highly recommended. Default location: data/seq/<project-name>/annotation

If either of the above are changed:
▸ If the annotation has already been loaded, Reload Annotation.
▸ If A&S has been previously run, re-run A&S (the existing alignments files will be reused).

▸ If this is changed after A&S, the alignment files need to be removed with Clear pair and A&S run again.

Order against
For draft contig sets, this allows you to order them using synteny to one of the other projects. See Ordering details.

▸ If the draft has been aligned to the Order against sequence, but this parameter was not set, it can be set and the A&S run with the existing alignment files.

  data/seq/<project-name>/params

Any parameter not the defaults will be shown on the View popup.

The params file parameters are shown on the Parameters window. These can only be viewed and changed using symap (not viewSymap).

2.g Annotation

   ID=gene-AT1G01010;Name=NAC001;ID=rna-NM_099983.2;product=NAC domain containing protein 1

If there are many different keywords in the attribute list, this causes too many columns in the Queries table. This can be reduced by one (or both) of the following:

Anno key count: If there are many different keywords in the attribute list, set this count N to filter out all keywords with <N occurrences. The Anno Key Count can be modified at any time using symap (not viewSymap).

Anno keywords: The keyword=value pairs to be saved for each gene can be limited by listing the desired keywords separated by commas. Using this approach, it will also reduce annotation description per gene in the 2D display. For example, if the string "ID, product" was entered, the Name=value would not be part of any gene annotation. This must be set on Load.

3.a Overview

Select a pair cell in the Available Syntenies table followed by the Parameters button, which will popup the window shown on the right.

If a ✔ or A in pair cell exists, and the parameters are changed, do the following: Alignment Select Clear Pair to remove the existing alignments, then Selected Pair (ReDo). Cluster Hits or Synteny Use Selected Pair (ReDo) directly; the existing alignments for the pair will be used.   The parameters are described in the following three sections. The most important parameter is for Algo2; the Intergenic parameter must be increased for conserved genomes.

Algorithm 2 (new, last update v5.4.8, abbreviated Algo2):
Pros This is a new algorithm that has explicit knowledge of gene pairs and their exon-intron structure, and shows all gene pairs with hits unless filtered by the parameters (and a few internal filters). It has better filtering for small intergenic and intron-only hits.
Cons It does not perform self-synteny or take NUCmer files as input. It does not work when a given chromosome is split over multiple MUMmer files (this will not happen when SyMAP generates the MUMmer files).
Parameters The parameters generally will need adjusting. However, this gives more control over what gene pairs are shown and can surpress the minor intergenic hits. See Hints below the parameter explanation.

Algorithm 1 is the default for now; this is because Algorithm 2 generally requires some experimenting with the parameters whereas Algorithm 1 does not.

Hint for Algorithm 1: Increasing the Top N parameter can cause too many hits and reduce synteny. Decreasing it can remove more gene-pair hits. Hence, try Algorithm 2 if you want more gene pairs.

Hint for Algorithm 2: On the output to the terminal, if any chromosome pair shows over 10,000 hits, the parameters probably need to be made more stringent; too many hits (and piles) confuses the synteny algorithm, which results in synteny blocks not being found; it also results in very long execution time. For highly similar genomes, it is necessary to increase the Intergenic parameter, e.g. >1000; you may also need to increase the others parameters and uncheck EI and En to reduce hits. For distant genomes, decrease the parameters. Suggestion: for large genomes, experiment with the parameters on just one pair of the chromosomes.

Piles of hits: The below image shows a pile of hits on the left that link to repetitive genes on the right. The left image shows a pile of hits in an intergenic region to multiple other regions. There are MANY occurrence of repeats like this in the MUMmer file, which is why these piles must be filtered; if they are not, the synteny algorithm does not perform well.

Running with ./symap -s provides additional output for both algorithms.

Wrong orientation

6,736 Both Genes - cluster strands differ from gene (Multi 1,425, Single 5,311)

You can view them in the Query where the Hit St column will be "=" but the gene Gst are not equal, or the other way around.

Selecting Block Merge may be beneficial when there are many small blocks. Below shows an example where the first image does not have merged blocks and the second does (the blue dots are hits that belong to the block).

  data/seq_results/<proj1-to-proj2>/params

The file parameters are shown on the pair Parameter window. These can only be viewed and changed using symap (not viewSymap).

Any parameter not the default will be shown on the Summary page.

BEWARE: If you run A&S, then change the PROmer or NUCmer settings, but forget to Clear Pair before running A&S again, the parameters on the Summary page will be wrong (SyMAP does not check for this situation).

3.f MUMmer parameters

To see the parameters for the default MUMmer V3 on MacOS, from the symap directory:

./ext/mummer/mac/mummer -h
./ext/mummer/mac/promer -h
./ext/mummer/mac/nucmer -h

./ext/mummer/linux/mummer -h
./ext/mummer/linux/promer -h
./ext/mummer/linux/nucmer -h

./ext/mummer4/m4/bin/mummer -h
./ext/mummer4/m4/bin/promer -h
./ext/mummer4/m4/bin/nucmer -h

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