Mega

Briefly described about MEGA by using some examples

1. Prepared By Nabajit Roy M.Sc 2 nd Semester Dept. of Life Science & Bioinformatics AUS

2. INTRODUCTION DOCS AND EXAMPLE DATA FILES MEGA INPUT FILES RUNNING MEGA MEGA-PROTO (ANALYSIS PROTOTYPER) USING MEGA-PROTO SEQUENCE ALIGNMENT CONSTRUCTION DATA HANDLING SEQUENCE ALIGNMENT SYSTEM IN MEGA ALGORITHM USED BEHIND MEGA SEQUENCE ALIGNMENT MAJOR MEGA VERSIONS AND THEIR RELEASED YEAR METHODS USED FOR PHYLOGENETIC TREE CONSTRUCTION PHYLOGENY ANALYSIS USING MEGA CONTENTs

3. MEGA (Molecular Evolutionary Genetics Analysis) is an integrated suite of tools for statistics-based comparative analysis of molecular sequence data based on evolutionary principles. MEGA is used by biologists for reconstructing the evolutionary histories of species and inferring the extent and nature of selective forces shaping the evolution of genes and species . MEGA has 2 components •MEGA-Proto, an analysis prototyper that is used for generating analysis options files which tell mega which analysis to run and which options to use. On Windows it is launched from the start menu. On Linux it is launched from a terminal using the megaproto command. On Mac it is launched from the Applications folder . •A command-line executable that performs all calculations. This executable is launched from a terminal using the mega command. INTRODUCTION

4. The installers for MEGA-CC copy doc files and example data files to OS-specific locations :: For Windows users - %HOMEPATH%\Documents\ megacc :: For Linux users - / usr /share/ megacc :: For Mac users - ~/Documents/ megacc For Linux and Mac users a man page is included with MEGA-CC. From a terminal: :: man megacc The doc files and example data files are also available from the mega website: :: http :// www.megasoftware.net/webhelp/helpfile.htm :: http :// www.megasoftware.net/examples.php DOCS AND EXAMPLE DATA FILES

5. MEGA Analysis Options file •Specifies the calculation and desired settings. •Created using MEGA-Proto. •Has a . mao file extension . Data file (one of the following) •Multiple sequence alignment in MEGA or Fasta format. •Distance matrix in MEGA format. •Unaligned sequences in Fasta format (for alignment only ). Newick tree file (required for some analyses) Calibrations file (for timetree analysis – but it’s optional) Groups file (optional ). MEGA INPUT FILES

6. In general, two output files are produced 1.Calculation-specific results file ( Newick file, distance matrix ,…). 2.A summary file with additional info (likelihood, SBL,…). •Some analyses produce additional output (bootstrap consensus tree, csv files, etc …). Output directory/filename •Default is the same location as the input data file. •Specify an output directory and/or file name using -o option. •If no output filename is specified, MEGA-CC will assign a unique name . Errors/warnings •If MEGA-CC produces any errors or warnings, they will be logged in the the summary file.

7. Easiest to run using command-line or batch scripts: • megacc –a settings.mao –d alignment.meg –o outFile Can also be run using custom scripts (Perl, Python, …): •exec(‘ megacc –a options.mao –d alignment.meg –o outFile ’); Integrated File Iterator system can process multiple files without the need for using scripts (see Demo2 below ) In addition, other applications can launch MEGA-CC: •status = CreateProcess (“path/to/ megacc …”); To see a list of available command options, call megacc from a command-line prompt with the –h flag (Unix users can view the man page). RUNNING MEGA

8. • Has the same look and feel as the GUI edition of MEGA. • Produces MEGA Analysis Options files. •Has no computational capabilities. MEGA-PROTO (ANALYSIS PROTOTYPER)

9. 1 . Select input data type. •Nucleotide (non-coding) •Nucleotide (coding) •Protein (amino-acid) •Distance matrix (MEGA format) 2 . Select analysis from menu. 3 . Adjust analysis settings. 4 . Click Save Settings... and save the MEGA Analysis Options (*. mao ) file to your computer. USING MEGA-PROTO

10. •The following example demonstrates how to create a timetree using MEGA-Proto and MEGA-CC DEMO 1

11. Alignment editor Multiple sequence alignment Sequencer (trace) file editor-viewer Integrated web browser, sequence fetching Fig. – Multiple sequence alignment SEQUENCE ALIGNMENT CONSTRUCTION

12. Handling ambiguous states: R, Y, T, etc . Extended MEGA format to save all data attributes. Importing data from other formats: Clustal , Nexus, etc . Data explorers Visual specification of domains-groups DATA HANDLING

13. GENETIC CODE TABLE SECTION Add-edit user defined tables Compute statistical attributes of a code table Include all known code tables REAL-TIME CAPTION EXPERT ENGINE Generate captions for distance matrices, phylogenies, tests, alignments Copy captions to external programs

14. SEQUENCE DATA VIEWER Data export Highlighting Statistical quantities estimation MCL-BASED ESTIMATION OF NUCLEOTIDE SUBSTITUTION PATTERNS 4x4 rate matrix Transition- transversion rate ratios: k1, k2 Transition- transversion rate bias: R

15. Pair wise sequence alignment Multiple sequence alignment Local alignment Global alignment Sequence alignment system in Mega

16. Smith waterman algorithm- This algorithm is basically used for local alignment where sequences are alignned in such a way that the sequecne tries to find complete resmblance with other one. Needleman wunsch algorithm- This algorithm is used for global alignment of sequences where sequences seats with respect to each other such that they show conserved and mutated regions. Algorithm used behind Mega sequence alignment

17. 1.0 1993 1.1 1994 2.0 2000 2.1 2001 3.0 2004 4.0 2006 4.1 2008 5.0 2011 5.1 Oct 2012 5.2 April 2013 6.0 December 2013 7.0 January 2016 10.0 (X) 2018 Major Mega versions and their released year

18. Maximum likelihood method Neighborhood joining method Maximum percimony method METHODS USED FOR PHYLOGENETIC TREE CONSTRUCTION

19. Sequences are downloaded from NCBI. Saved in word file. Now using MEGA sequences are aligned by alignment tool. After complete alignment the phylogeny option is chosen and as per need the tree is made . After tree formation for proper study an outgroup is taken in consideration depending on literature available. The length of each nodes and internodes taken in consideration so to analyze evolutionary relationship. Phylogeny analysis using Mega

20. Kumar, Sudhir , Glen Stecher , and Koichiro Tamura. "MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets."  Molecular biology and evolution  33.7 (2016): 1870-1874 . Tamura , Koichiro , et al. "MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0."  Molecular biology and evolution  24.8 (2007): 1596-1599 . Tamura, Koichiro , et al. "MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods."  Molecular biology and evolution  28.10 (2011): 2731-2739 . https://en.wikipedia.org/wiki/MEGA,_ Molecular_Evolutionary_Genetics_Analysis https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562624 / https://www.ncbi.nlm.nih.gov/pubmed/27004904 references