Network 5.0.0.0 User Guide

Version date: 24 December 2015 Copyright 2015 Fluxus Technology Ltd. All rights reserved.

Legal Disclaimer :

This user guide shall not be interpreted as a warranty of any kind. Use of the software is subject to the terms under www.fluxus-engineering.com/network_terms.htm

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Preface Today marks the release of a major new version of the free Network software. Network 5.0.0.0 has been programmed with a new commercial software development environment instead of the old free development environment that we used up to version 4.6.1.4. Our main reason was to fix all the minor display problems of the past years, as our free development environment just did not support the countless displays. Secondly, users increasingly ran into memory error messages despite sufficient computer memory, which turned out to be caused by bugs in the the way that the old development environment compiled our Network code. We started work about 2 years ago, and it has taken us longer than anticipated to move from Network 4.6.1.3 to Network 5.0.0.0. Indeed, some menu items in Network 5.0.0.0 are still not functional and will be fixed in the next months:

1. Time estimates 2. Tools / Mismatch distribution calculations

If Time estimates and mismatch distributions are of interest to you, please use them in Network 4.6.1.4. As a collateral casualty, PDF reports were removed from Network 5.0.0.0. PDF output does not work properly with our new commercial software development environment. If you need PDF reports, please consider the use of the non-free Network Publisher. Because the transition from Network 4.6.1.3 to Network 5.0.0.0 is not yet complete, this user guide is not fully updated. All screen snapshots still refer to the old Network version. Looking back, Network has come a long way since we released the first DOS version in January 2000. The network reduction strategies described in this user guide are even more relevant today than in 2000, when data sizes were generally smaller and hairballs were not yet a general problem. If you run into a problem, we hope that this updated user guide will continue to help you to get a meaningful network out of your data. Michael Forster, 24 December 2015

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Table of Contents Preface ...................................................................................................................................2 1. Overview............................................................................................................................5 1.1 Scope of application ........................................................................................................5 1.2 Network building options ................................................................................................5 1.3 Further complexity reduction options...............................................................................5 1.4 Complementary options...................................................................................................5 2. Work Flow .........................................................................................................................6 2.1 Overview of the general work flow and the RM-MJ work flow.........................................6 2.1.1 Variable data .................................................................................................................8 2.1.2 Preparation of variable data sets for Network.................................................................9 2.1.3 Weights .......................................................................................................................12 2.1.4 Frequency....................................................................................................................16 2.1.5 Epsilon (in MJ), Connection Cost / Greedy FHP (in MJ) / MJ square option................17 2.1.6 Reduction threshold r and out file option (in RM network option)................................20 2.1.7 MP option to clean up networks...................................................................................22 2.1.8 Star Contraction option: Use for network simplification, or for identification of

population expansion events........................................................................................24 2.1.9 "Frequency>1" Criterion for networks with large number of taxa ................................26 2.1.10 RM-MJ network calculation for reduced complexity.................................................27 2.2 DNA nucleotide sequence data .......................................................................................28 2.2.1 Data entry....................................................................................................................28 2.2.2 Network calculation using the MJ algorithm with optional external rooting .................29 2.2.3 Discussing, analysing, and interpreting network results (MJ and RM)..........................31 2.2.4 Graphical layout of results ...........................................................................................33 2.2.4.1 Node and pie chart colouring in Network Publisher 2.0.0.0.......................................34 2.2.5 Verification using the RM option.................................................................................36 2.3 RNA nucleotide sequence data .......................................................................................38 2.3.1 Data entry....................................................................................................................38 2.4 Amino acid nucleotide sequence data .............................................................................39 2.4.1 Data entry....................................................................................................................39 2.4.2 Network calculation, analysis, interpretation, and graphics ..........................................40 2.5 STR data (short tandem repeat, microsatellite data) ........................................................41 2.5.1 Data entry....................................................................................................................41 2.5.2 Network calculation, analysis, interpretation, and graphics ..........................................42 2.6 Endonuclease data (RFLP, restriction fragment length data) ...........................................43 2.6.1 Data entry....................................................................................................................43 2.6.2 Network calculation, analysis, interpretation, and graphics ..........................................44

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2.7 Binary data .....................................................................................................................45 2.7.1 Data entry....................................................................................................................45 2.7.2 Network calculation, analysis, interpretation, and graphics ..........................................45 2.8 Time estimates ...............................................................................................................46 2.8.1 Calibration of network mutation rate with a known event ............................................46 2.8.2 Age estimation of a node in the network ......................................................................48 3. Software Limits in Network 5.0.0.0..................................................................................50 4. Network 5.0.0.0.: Present and Future................................................................................51 5. Feedback: Bug Reports and Enhancement Requests .........................................................52 6. Updates to the Network 4.6.1.1 User Guide.................................................................53 7. Updates to the Network 4.6.1.0 User Guide.................................................................53 8. Updates to the Network 4.6.0.0 User Guide.................................................................53 9. Updates to Network 4.5.1.6 User Guide (Compared to Network 4.5.1.0 User

Guide of 27 December 2008) ......................................................................................53 10. Updates to Network 4.5.1.0. User Guide (compared to Network 4.5.0.1 User

Guide of 24 June 2008) ...............................................................................................54 11. Updates to Network 4.5.0.1 User Guide (compared to Network 4.5.0.0 User Guide

of 31 December 2007).................................................................................................54 12. Updates to Network 4.5.0.0 User Guide (compared to Network 4.2.0.1 User Guide

of 19 September 2007) ................................................................................................54 13. Updates to Network 4.2.0.1 User Guide (compared to 3 April 2007) ...........................55

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1. Overview

1.1 Scope of application Network is used to reconstruct phylogenetic networks and trees, infer ancestral types and potential types, evolutionary branchings and variants, and to estimate datings.

The algorithms are designed for non-recombining bio-molecules. Successful applications include mtDNA, Y-STR, amino acid, RNA, virus DNA, bacterium DNA, some effectively non-recombining autosomal DNA, and non-biomolecule data such as linguistic data. By contrast, recombining bio-molecules will deliver high-dimensional networks which will be difficult to interpret. Work flow including data preparation and interpretation of results is described in detail in the next chapters.

1.2 Network building options The