Free Phylogenetic Network Software | Information | Troubleshooting and FAQs

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Documentation: A new user guide (pdf, 2MB) is available on the Network download page.

1. Test version expiry

Network will expire at the end of the calendar year. It is intended that new versions will continue to be provided after this date.

2a. Compatibility between Windows and DOS versions

Network4.x for Windows accepts all input and output files generated by Network2.x for DOS. Reverse compatibility (from 4.x back to 2.x) also works, with the following exceptions:

(a) One file name ending has been renamed: *.mat (version 2.x) is now *.rmf (version 4.x)
(b) Deletion coding (dash) is available in 4.x, but not in 2.x.
(c) Branch lengths are limited to 25 mutations in 2.0.
(d) File names may be up to 255 characters long in version 4.x , but only 8 characters in version 2.x.
(e) Network 4.x displays text on Japanese computers, whereas Network 2.x does not.

2b. Screen display adjustment

Normally Network 4.x adapts its Windows to your screen size. However, sometimes scrollbars may appear.
Remedy: To avoid scrollbars, make sure that font size, icon size, dpi etc. of your computer are set to default values (100%). For example for English Windows 98 versions, the menus are:
Start>Settings>Display Properties>Effects> and
Start>Settings>Display Properties>Settings>Advanced>General>Font Size.

Display distortion problems may happen on some computers (typically laptops with very high resolution displays) and can by resolved by changing the DPI-Settings to "Normal (96DPI)" as follows: a) Right-click your Desktop, b) Display Properties, c) Settings, d) Advanced, e) General, f) DPI-Settings --> changed these to "Normal (96DPI)", g) re-boot Windows. You may find that the Windows fonts are now too small for you. To rectify this, change the display resolution to 800x600 or 1024x768 as follows: a) Right-click your Desktop, b) Display Properties, c) Settings --> change the resolution.

3. Choice of data format

The choice of data formats is explained step by step within the Network 4.x software.

4. Data entry problems

Small data sets are best re-entered manually. The procedure is explained step by step within Network.

For importing sequences into Network, the DNA Alignment software is strongly recommended. This avoids file formatting problems, which can lead to unnoticed errors and/or cost a lot of wasted time. Network results will normally be wrong when non-aligned or badly formatted sequence data are used ("garbage in, garbage out" principle).

The sequential Nexus (*.nex) and Phylip (*.phy) formats are not generally recommended for importing sequences into Network. These formats were originally included into Network at the wish of specific professional users. However, the general use of these formats with Network can be disastrously error-prone, leading to aborts with odd error messages or to wrong network results. New users who nevertheless wish to try out the *.nex or *.phy imports should be aware of the following points: a) the sequence data in *.nex/*.phy files must be properly aligned, b) aligned data in *.rdf format from the DNA Alignment software is preferable, c) the *.nex/*.phy file formats must be strict (not all software exports strict Nexus or Phylip files), d) the *.nex/*.phy files should not contain empty lines, d) the *.nex/*.phy files should not contain tabstops, e) the formatting of text within each line must be correct (e.g. sufficient leading spaces), f) all line breaks must be in the correct places, g) the *.nex/*.phy file headers must contain the correct number and lengths of sequences, h) in the *.nex/*.phy files the sequence lengths must be correct in each sequence definition.

Alternatively, users may wish to create "home-made" files using for example Editor/Notepad. This is tedious and error-prone and we do not recommend it for the general user, except for the simple *.ych format. Experienced users may create home-made files by consulting the examples in the Network download. Five acceptable entry formats are: multistate *.rdf, binary *.rdf, *.ych, *.ami and *.tor. Taxon names in any file format should not be longer than 6 characters, and each taxon name MUST be unique in the file. Nucleotide, RFLP and STR names must not be longer than 5 characters in any of the three file formats. Sequence length (number of characters) must not be longer than 1000 positions in any file format. For Roehl data format (*.rdf), consult the examples included in the download. For Y STR format (*.ych) note that there is a limit of 100 STR loci. Furthermore, beware that each STR entered in ych-format will be broken up into several characters when Network4.x converts it into rdf-format, possibly exceeding the limit of 1000 positions per sequence; a trial run is advisable. Torroni RFLP format (*.tor) is the simplest to generate; consult the self-explanatory *.tor example file in the download. A maximum of 35 sites per sequence is permitted. Users who construct such "home-made" files often struggle with formatting errors. A single format error may cause Network to produce artefacts without an error message. Beware that MS Word and Windows Wordpad are unsuitable for editing your data, because they can insert/delete spaces unpredictably. This causes Network to produce artefacts. It is safe to use the Windows text editor Notepad (called Editor on some non-US-language Windows versions). In Network for Windows, the file format is not recognised if the appropriate file ending (*.rdf or *.ych or *.tor) is missing. Network for DOS is flexible in this respect. You can rename the file endings in Windows Explorer, if the file endings are activated. To activate file endings in Windows 98, go to Windows Explorer>View>Folder Options>View>Hide file extensions for known file types (deactivate). Files generate error messages if there are empty lines at the end of a file (often the case when converting from Excel). Files generate error messages if values are placed in quotation marks (often the case when converting from Excel).

5. Example file discrepancies

Example files for Torroni RFLP format (east Asian mtDNA RFLPs), Y STR format (Amerind Y STRs), Roehl data format (Nuu Chah Nulth mtDNA control region), and amino acid format (primate AB0 enzyme data) are included in the download. All four analyses are discussed in the literature: consult Fig 3 in Forster et al. (2001) for the east Asians; Fig 4 in Forster et al. (2000) for the Amerinds; Fig 7 in Bandelt et al. (1995) for the Nuu Chah Nulth; and Fig 7 in Bandelt et al. (1999) for the primates.

Three of these publications contain errors which the attentive user will discover when recalculating the examples: In the Forster et al. (2001) paper on pages 1870/1871, the value for delta was set to 5 (rather than 3), and the first round of star contraction reduced the data from 245 types to 113 (rather than 115) types. In the Bandelt et al. (1999) Figure 7, additional reticulations involving amino acid position 174 were erroneously omitted due to a previous MJ bug which sometimes occurred when epsilon>0. In the Forster et al. (2000) Figure 4, the epsilon value was set to zero rather than to 0.6, as explained in the subsequently published erratum. These errors do not influence any other numerical value or result presented in the papers.

Weights/epsilon discrepancy between the publications and current versions of the software: In the Network rdf/STR editor, weights are integer numbers between 0 and 99, the default is 10. To re-enter the example data from the above publications, enter a weight-value 10x larger than in the publication, and an epsilon value 10x larger than in the publication; for example enter a weight of 10 instead of 1.0, and enter the epsilon value as 6 instead of 0.6.

6. Network calculation

If your data are binary and you expect branches which are more than a few mutations long (you can get an idea by displaying the mismatch distribution available in the File menu), then preferentially use the RM algorithm, otherwise resort to the MJ algorithm.

The first thing you should do with any data file is to call up the Change Weights option within RM or MJ to check whether all your characters or nucleotide positions were entered and weighted correctly. We suggest running an initial analysis with the default settings, that is, r set to 2 if you choose RM, or epsilon set to zero if you choose MJ.

If the networks turn out to be clean (i.e. treelike, and without large cycles), you should experiment with slightly higher settings to visualise the extent of homoplasy (potentially due to recurrent mutations, sequence errors, recombination etc.).

If on the other hand the initial network is messy (high-dimensional cubes) or contains an empty cycle larger than a rectangle (only in MJ networks), then something is amiss (recurrent mutations, sequence errors, recombination). To explore or overcome the problem, activate the frequency>1 option before running the algorithms; this option will select only those sequences confirmed at least twice in the data set. If the network is still messy, you can investigate whether this may be due to a few rapidly mutating characters by consulting the statistics option. These characters are candidates for downweighting before running another analysis. Weighting may be particularly relevant for STRs: the program internally codes the entered STRs assuming a single-repeat mutation mechanism. If this is known to be unrealistic for a given STR, the offending STR should be dealt with by downweighting it as a whole, or by differentially weighting its length transitions (labeled with a, b, c...). In general, weights often are most effective when chosen conservatively, e.g. a known tenfold higher mutation rate for a nucleotide position should be translated into a much less extreme than tenfold lower weight setting in the network calculation.

If despite these efforts the network still contains many high-dimensional features, then (for binary data files), RM and MJ can be applied sequentially. First, use RM to generate a *.rmf file (the *.out file will also be generated but is of no consequence here); then, apply MJ to the *.rmf file.

7. Large data sets

If your data set contains hundreds of sequences and the corresponding network is consequently difficult to visualise, use the star contraction option prior to the phylogenetic analysis. The star contraction option reduces large data sets to smaller data sets by identifying and contracting any starlike phylogenetic cluster into one ancestral type. The reduced data set can then be run in a phylogenetic algorithm (either our network methods or other tree-building methods) to produce a simplified skeleton phylogeny. In the graphical display, the algorithm remembers which sequences were contracted. An example star contraction analysis is included in the download and is discussed in Forster et al. (2001). Note that the publication contains some typographical errors (which do not influence the presented values or conclusions) on pages 1870/1871: for the Asian analysis the value for delta is set to 5, and the first round of star contraction reduces the data from 245 sequence types to 113 sequence types.

8. Network graphics in better quality for publication or printing

The graphics file formats *.bmp and *.pdf produced by the free Network software contain bitmaps which may be of limited value for publication. The Network Publisher software can produce vector graphics formats (*.wmf and *.emf) which havepublication-quality resolution. As a further benefit, the vector objects (network nodes, links, pies, names) can be selectively edited, moved or deleted after importing the files into photo/publishing or office software such as Adobe CS4, Corel Draw, and Microsoft Office.

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