Briefly described about MEGA by using some examples

1. Prepared By
Nabajit Roy
M.Sc 2nd 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%Documentsmegacc
:: 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 MEGAAnalysis 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. 1. Pair wise sequence alignment
2. Multiple sequence alignment
3. Local alignment
4. 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. 1. Maximum likelihood method
2. Neighborhood joining method
3. Maximum percimony method
METHODS USED FOR PHYLOGENETIC
TREE CONSTRUCTION

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

20. 1. 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.
2. Tamura, Koichiro, et al. "MEGA4: molecular evolutionary genetics analysis (MEGA) software
version 4.0." Molecular biology and evolution 24.8 (2007): 1596-1599.
3. 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.
4. https://en.wikipedia.org/wiki/MEGA,_Molecular_Evolutionary_Genetics_Analysis
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562624/
6. https://www.ncbi.nlm.nih.gov/pubmed/27004904
references