The 'mfold' software is a tool for nucleic acid folding and hybridization prediction. It was developed by M. Zuker in the late 1980s for RNA folding and improved for DNA folding in 1996. mfold uses nearest neighbor energy rules.

Tool home page: mfold.org. Similar results to those produced by UGENE can be obtained using the mfold web server (DNA prediction, RNA prediction) or using the OligoAnalyzer tool. For complete documentation on the tool, see a) the website, b) the article, c) the source code in the corresponding folder.

UGENE uses Ghostscript to convert PS files to PNG and PDF.

ToC

Prerequisites

For mfold to work correctly, the path to the temporary directory must not contain spaces, non-printable characters and non-ASCII characters (Unicode characters such as emoji, Cyrillic, Chinese family of scripts, etc.). In this case, you need to change the path to the temporary directory in the appropriate setting.

UGENE itself must be installed to a path that does not contain non-printable characters or non-ASCII characters (Unicode characters). If installed incorrectly, a corresponding error while running the mfold task will be displayed.

Open the dialog

mfold only works with DNA or RNA sequences. You can trigger the dialog form
by selecting the appropriate Sequence View Global action 
or through the Main Menu→Actions→Analyze                    
or through the Sequence Context Menu→Analyze              

This is what the mfold dialog looks like

Input parameters

The tool is passed explicit and implicit parameters. Explicit parameters are set in the dialog, implicit parameters are taken from the sequence context.

Algorithm settings

For complete settings details, see the documentation on the mfold website or documentation in source.

This is the Settings section of the mfold settings tab.

Display settings

Corresponds to the Extended settings section on the main tab.

Region settings

Specifies the part of the sequence that will be analyzed. The tool will only work with this piece of the sequence in isolation from the whole sequence.

You can select the entire sequence, a custom region, or a selected region if there was one. The region must be less than 3000 bases. It is acceptable to select a small region (<10bp), but there will almost certainly be no folding there.

If several regions have been selected in a sequence (using the GenBank format), then the dialog will only accept the first of them. The remaining selected regions are ignored. But there is an exception: if the sequence is marked circular and the region passing through the end/beginning is selected, then such a region is considered as a whole region and mfold will be launched on it as on an ordinary piece of the sequence (2 parts of the region – one that goes to end and another that starts from the beginning of the sequence – will be combined into one sequence and analyzed). This is the only case where the start of a region can be greater than the end:

                                                                        
A linear sequence is marked with the symbol  and for it the region 70..50 is invalid. A circular sequence is marked with the symbol  and for it the region 70..50 is valid.
The length of the region in this case 70..50 depends on the sequence length.

Output settings

UGENE&Ghostscript settings in the "Output" tab.

C:.
└───path
    └───to
        └───sequence
            │   my_sequence.fa
            │
            └───mfold
                └───2024.03.19_16-31-34
                        inp.fa_1.pdf
                        inp.fa_1.png
                        inp.fa_2.pdf
                        inp.fa_2.png
                        inp.fa_3.pdf
                        inp.fa_3.png
                        out.html

Internal parameters

Settings that the user cannot explicitly influence.

1. Molecule Type (DNA/RNA). mfold gets sequence information from command line arguments. Information about what type will be passed to the tool can be seen next to the sequence name
   
2. Molecule Topology (linear/circular). mfold gets sequence information from command line arguments. If the sequence is marked as circular, then mfold will work with it as a circular one. Otherwise, as with linear one.
   Whether the sequence is linear/circular is described here.
   Part of a circular sequence will also be considered a circular sequence. If you want to change this behavior, uncheck "Mark sequence as circular" and then call the mfold dialog.
   
3. DPI of images in UGENE. Ghostscript has a default DPI of 72. Therefore, this value is used for images for the internal UGENE report and this value cannot be changed.

Output data

There are 3 types of output from the mfold task in UGENE:

1. Report displayed inside UGENE.
2. HTML report with images saved on disk.
3. Temporary data (for advanced users).

UGENE report

Example of a report on Windows (appearance may vary slightly between systems):

1. Status, Time, … At the beginning there is information about the run: information about the task, settings for launch.
2. Output HTML report. Clickable link to the output HTML report. More about this report in the next section.
3. Found structures. List of found structures. The structure is characterized by free energy. When clicked, UGENE will scroll the window to the desired structure.
4. Structure table. For each found structure, the table contains:
   1. Thermodynamic details: free energy, enthalpy, entropy and an estimated T_m.
   2. Loop Free-Energy Decomposition: the entire decomposition of the particular folding into loops and stacks, together with their free energies and closing base pairs. Consecutive runs of base pairs are summarized as helices.
   3. Image. Displays of nucleic acid secondary structure. At the bottom of the image there is information identifying it: free energy, name of the input sequence and analyzed region. It has the above-mentioned fixed DPI of 72.
5. mfold log. Information about launching the external mfold tool: launch command and log.
   
   

HTML report

A single HTML report and PNG+PDF for each structure found are saved in the output folder. The HTML report displays these images, PDFs and detailed information about the launch in an easy-to-view form.
This version of the report also contains:

1. Launch information.
2. List of found structures with links to them.
3. Information about each structure. The Loop Free-Energy Decomposition is located under the "Show structure detailed info" arrow.
   The quality of the images in the report depends on the DPI setting in the dialog. When you click on the picture, a PDF file with a structure image opens in a new tab, identical to the picture image, but allows you to zoom without losing quality.
   More details are described in the Structure table subsection of the UGENE report section.
4. mfold log.

This report is portable, i.e., if the directory is completely copied (HTML+all PNGs+all PDFs), the copied data can be re-opened in the browser without loss of functionality. Folders can be deleted/renamed; it will not affect the operation of UGENE.

Temporary output

mfold produces many files. Not all of them are displayed in the reports. However, while the current UGENE session is open, you can see what other files mfold produces.

In the Log corresponding to the [Algorithms][DETAILS] subcategories, you can see the mfold launch command. It depends on where the UGENE temporary directory is set. For example, it might look like this

"C:\Program Files\Unipro UGENE\tools\mfold\mfold.bat" SEQ=C:\tmp\ugene_tmp\p14120\mfold\d_0\inp.fa RUN_TYPE=html NA=DNA LC=circular T=37 P=5 NA_CONC=1 MG_CONC=0 MAX=100 ROT_ANG=0

In this case, all temporary output files will be located in C:\tmp\ugene_tmp\p14120\mfold\d_0. For information about the purpose and format of this data, please read the article.

Tool comparison

As mentioned earlier, the mfold tool can also be launched from the native site or from the IDT platform.

https://en.wikipedia.org/wiki/List_of_RNA_structure_prediction_software

References

1. Article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC169194/
2. mfold home http://www.unafold.org/
3. mfold DNA prediction http://www.unafold.org/mfold/applications/dna-folding-form.php
4. mfold RNA prediction http://www.unafold.org/mfold/applications/rna-folding-form.php
5. mfold sources http://www.unafold.org/mfold/software/download-mfold.php
6. mfold settings documentation http://www.unafold.org/mfold/documentation/mfold-documentation.php
7. Viewing launch results http://www.unafold.org/cgi-bin/view-folds.cgi
8. Forum with mfold authors http://www.unafold.org/forum/
9. mfold references http://www.unafold.org/mfold/documentation/mfold-references.php
10. Implementation of mfold by IDT company (OligoAnalyzer) https://www.idtdna.com/calc/analyzer