Home

User Manual for MEGAN V3.8 - HIGH THROUGHPUT SEQUENCING

1. 16 Open File 9 Open NCBI Web Page 14 16 Open Recent 6 9 Optimize View For Large Data Sets 22 Options 13 19 Options gt Apply Ignore Use Changes 19 Options gt Change LCA Parameters 13 24 Options gt Chart GO 23 Options Collapse 19 Options Expand 19 Options Extract Reads By GOs 22 23 Options Highlight Incident Nodes of Selected Edges 22 24 Options Highlight Paths of Selected Nodes 22 Options gt Ignore Hit 19 Options gt Inspect 14 Options gt Inspect GO 22 23 Options List COGs 14 Options List Microbial Attributes 14 Options List Summary 14 Options Open NCBI Web Page 14 Options Select Subgraph 22 Options Set Number of Reads 5 14 Options Show GO Term in List 22 24 Options Show Read 19 Options Show Taxon 18 19 Options Taxon Disabling 8 13 Options Taxon Disabling Disable Selected 13 Options Taxon Disabling Enable All 13 Options Taxon Disabling Enable Selected 13 Options Taxon Disabling List Disabled 14 Options Use All Hits 19 Options Use Hit 19 Order 12 14 Page Setup 10 21 Parameters 24 27 Parse taxon names 18 Paste 11 19 PDF 28 Phyla 12 14 Plant GO Slim 23 PNG 28 popup menu 16 23 24 Preferences 11 22 preferences 32 Preserve existing files 25 Print 10 16 18 21 Prokaryotic Subset 23 Properties 11 properties file 32 Quit 11 16 read file 26 r
2. Fit Content 23 Font Size 12 font change 24 Format 11 24 27 Forward 20 From File 20 From Previous Window 12 Full Dataset 27 full dataset 28 Full View 23 Fully Contract 13 16 Fully Expand 13 16 Generic GO Slim 23 genome 3 GIF 28 GOA and Proteome GO Slim 23 ezipped BLAST files 28 38 Highlight Differences 13 Highlight Incident Nodes of Selected Edges 22 24 Highlight Paths of Selected Nodes 22 24 horizontal zoom 17 How to cite 3 15 Ignore Not Assigned and No Hits 25 Ignore Hit 19 Import 17 Import BLAST 5 9 26 Import CSV 10 31 Import wizard 17 Inspect 5 14 16 18 Inspect GO 22 23 Inspector 18 Inspector Window 15 18 Invert 12 iText 35 JFreeChart 35 JPEG 28 Kingdom 12 14 Labels Off 15 16 Labels On 15 16 Layout 12 Layout Labels 12 Layout Draw Circles 13 Layout Draw Heat Maps 13 Layout Draw Heat Maps 2 13 Layout Draw Leaves Only 13 Layout Draw Meters 13 Layout Draw Pie Charts 13 Layout Expand Contract 12 Layout gt Expand Contract gt Contract Horizon tal 12 16 Layout gt Expand Contract gt Contract Vertical 12 16 Layout gt Expand Contract gt Expand Horizon tal 12 16 Layout gt Expand Contract gt Expand Vertical 12 16 Layout Font Size 12 Layout Fully Contract 13 16 Layout Fully Expand 13 16 Layout Highlight Differences 13 Layout Layout
3. Preferences Show Colored Read Assign ment Table 22 Edit Preferences Show Legend 11 Edit Preferences Show Node Labels 22 Edit Preferences Synchronize GO Term Se lection 22 Edit Select All 19 Enable All 13 Enable Selected 13 enabling taxa 13 Enter a command 15 environmental samples 13 EPS 28 examples 5 Expand 19 Expand Horizontal 12 16 Expand Vertical 12 16 Expand Contract 12 Export 21 Export Graph View 21 Export Image 10 25 Export Read Assignment 21 Export Table View 21 Extract Reads by COG 10 Extract Reads By GOs 22 23 Extract Reads By Taxa 16 Extract Reads by Taxa 10 Extractor 25 Family 12 14 File 9 18 21 File Close 11 18 21 File Compare 10 25 File Export 21 File Export Graph View 21 File gt Export Image 10 25 File Export Read Assignment 21 File Export Table View 21 File Export Assignments 10 File Export Blast 10 File Export Reads 10 25 File gt Export gt Summary 10 File gt Extract Reads by COG 10 File gt Extract Reads by Taxa 10 File gt Import BLAST 5 9 26 File Import CSV 10 31 File New 9 File Open 6 9 16 File gt Open Recent 6 9 File Page Setup 10 21 File Print 10 16 18 21 File Properties 11 File gt Quit 11 16 File Save As 9 18 File Tools 11 Files pane 17 Find 5 11 16 19 22 23 Find Again 11 Find All 20 Find First 20 Find Next 20
4. This reduction greatly simplifies the problem of analyzing and navigating the large numbers of reads contained in typical metagenomic data sets 11 Main Window The Main window is used to display the taxonomy and to control the program via the main menus Initially at startup before reopening or creating a new RMA file the Main window displays the NCBI taxonomy By default the taxonomy is only drawn to its second level Parts of the taxonomy or the full taxonomy can be explored using the menu items of the window Once a data set has been read in the full NCBI taxonomy is replaced by the taxonomy that is induced by the data set The size of nodes indicates the number of reads that have been assigned to the nodes using the algorithm described in Section 9 Double clicking on a node will produce a textual report stating how many reads have been assigned to the corresponding taxon and how many reads have been assigned in total to the taxon and to any of the taxa below the given node in summary Subtrees can be collapsed and expanded as described below We now discuss all menus of the Main window 11 1 File Menu The File menu contains the following file related items e The File gt New item opens a new empty MEGAN window e The File Open item provides an Open File dialog to open one or more RMA files containing input data e The File Open Recent item can be used to re open a recently opened files The File Save As item can b
5. cluster of orthologous groups 7 COG 7 Collapse 14 16 19 Collapse Nodes at Level 14 Collapse Nodes at Taxonomical Level 14 Collapsed Nodes 20 Collapsed taxa 5 color change 24 Command syntax 15 command line 32 command line mode 32 commands 33 Compare 10 25 comparison 28 Content pane 17 ContentType 27 Contract Horizontal 12 16 Contract Vertical 12 16 control click 16 23 Convert text to graphics 25 Copy 11 19 Copy Edge Label 17 Copy GO ID s 22 23 Copy GO Name s 22 Copy GO Names 23 Copy Node Label 16 CreationDate 27 Creator 27 CSV file 31 Cut 11 19 Disable Selected 13 disabling taxa 13 Disclaimer 3 Draw Circles 13 Draw Heat Maps 13 Draw Heat Maps 2 13 Draw Leaves Only 13 Draw Meters 13 Draw Pie Charts 13 Edges 20 Edit 11 18 Edit Comparison Colors 11 Edit Edge Label 11 17 Edit Node Label 11 16 Edit Clear 19 Edit Copy 11 19 Edit gt Copy GO ID s 22 Edit Copy GO Name s 22 Edit Cut 11 19 37 Edit Edit Edge Label 11 Edit Edit Node Label 11 Edit gt Find 5 11 16 19 22 23 Edit Find Again 11 Edit Format 11 24 Edit Paste 11 19 Edit Preferences 11 22 Edit Preferences Antialiased Painting 22 Edit Preferences Edit Comparison Colors 11 Edit Preferences Node Coloring 22 Edit Preferences gt 0Optimize View For Large Data Sets 22 Edit Preferences Set Label Font Size 22 Edit
6. Labels 12 Layout Scale Nodes By Assigned 12 Layout Scale Nodes By Summarized 12 Layout Set Max Node Radius 12 Layout Zoom to Selection 13 LCA Parameters pane 17 Level 12 line width change 24 Linux 6 List COGs 14 List Disabled 14 List Microbial Attributes 14 16 List Summary 14 Load Accession Lookup 18 Load Synonyms File 18 MacOS 6 Main 9 Max number of matches per read 17 MEGAN 16 MEGAN file 27 MEGAN_macos_3 8 dmg 6 MEGAN_unix_3 8 sh 6 MEGAN_windows_3 8 exe 5 Message 24 Message Window 15 24 Messages 20 metagenome 3 metagenomics 3 Microbial Attributes Window 15 Min Score 24 Min Score Length 24 Min Support 24 MRJ Adapter 35 NCBI mapping file 32 NCBI taxonomy 7 NCBI tree file 32 NCBI NR 7 NCBI NT 7 New 9 Node Coloring 22 node size change 24 Node Inspect 5 18 Nodes 20 39 Nodes Collapse 16 Nodes Copy Edge Label 17 Nodes gt Copy GO ID s 23 Nodes Copy GO Names 23 Nodes gt Copy Node Label 16 Nodes Edit Edge Label 17 Nodes Edit Node Label 16 Nodes Extract Reads By Taxa 16 Nodes Highlight Paths of Selected Nodes 24 Nodes Inspect 16 Nodes gt Labels Off 16 Nodes gt Labels On 16 Nodes List Microbial Attributes 16 Nodes Open NCBI Web Page 16 Nodes Select Subgraph 23 Nodes gt Uncollapse 16 Nodes gt Uncollapse Subtree 16 None 11 Normalize over all reads 25 Open 6 9
7. Use Accession Lookup check box item is used to turn the use of accession lookup on and off Please note that identifying taxa using accession lookup is much slower than just using name parsing and thus should only be used when really needed The Load Synonyms File can be used to load a file of customized synonyms to help identify taxa e g human for homo sapiens Each line of a synonyms file should contain two strings separated by a tab the synonym followed by the taxon name The Use Synonyms check box item is used to turn use of Synonyms on and off 13 Inspector Window The Inspector Window can be used to inspect the alignments that are the basis of the assignment of reads to taxa It can be opened either using the Window Inspector Window menu item or by right clicking on a taxon and then selecting the Inspect popup item This window displays data hierarchically using a data tree The root node of this tree represents the current input file This window can only be opened when data has been loaded into the program Any taxon added to the window either by right clicking a taxon and then selecting the Inspect popup item in the main viewer or by using the Options gt Show Taxon item is shown at a second level below the root Clicking on such a taxon node will open a new level of nodes each read node representing a read that has been assigned to the named taxon Clicking on a read node will then open a new level of nodes each such read hit nod
8. and analyze a the result of a BLAST comparison of a set of reads against one or more reference databases typically using BLASTN BLASTX or BLASTP to compare against NCBI NT NCBI NR or genome specific databases The result of a such an analysis is an estimation of the taxonomical content species profile of the sample from which the reads were collected The program uses a number of different algorithms to place reads into the taxonomy by assigning each read to a taxon at some level in the NCBI hierarchy based on their hits to known sequences as recorded in the BLAST file MEGAN2 introduces many new functionalities including the ability to open multiple documents and to compute a comparative view of multiple datasets to extract reads from a set of FastA files by taxon to compute an analysis of COGs discovered in the dataset to use accession numbers to help identify reads and some basic charting capabilities as from version 3 0 onward MEGAN uses a binary format to save information rather than a text file For an example of its application see 4 where an early version of this software called GenomeTax onomyBrowser was used to analyze the taxonomical content of a collection of DNA reads sampled from a mammoth This document provides both an introduction and a reference manual for MEGAN 2 Getting Started This section describes how to get started First download an installer for the program from www ab informatik uni
9. clipboard e The Collapse item collapses hides the subtree below the selected node e The Uncollapse item un collapses the children of the selected node e The Uncollapse Subtree item un collapses the subtree below the selected node e The List Microbial Attributes produces a textual summary of the taxon represented by the selected node e The Extract Reads By Taxa stores the reads assigned to selected taxa into one or more FASTA files A dialog window allows the user to choose output directory as well as file names e The Labels On is used to make the label of a node visible e The Labels Off is used to make the label of a node invisible e The Open NCBI Web Page shows the NCBI taxonomy web page for the selected taxon The popup menu that is opened when an edge is right clicked on has the following items 16 e The Copy Edge Label copies the node label to the system clipboard e The Edit Edge Label opens a dialog to change the label of the selected edge If the shift key is pressed when using the popup menu for either an edge or a node then the chosen item is applied to all currently selected edges or nodes and not just to the one hit by the mouse clicks 11 11 Wheel Mouse and Special Keys Use of a wheel mouse is recommended for zooming of the Main window The default is vertical zoom For horizontal zoom additionally press the alt key To scroll the graph either press and drag the mouse using the right mouse button
10. completed when necessary To open a file print the taxonomical analysis and then close the file using the x option enter the following x open meganfile myfile rma update exportgraphics format PDF file myfile pdf quit Here is a summary of the commands available in command line mode Creating a new MEGAN project and reopening projects import blastfile name readfile name meganfile name maxmatches num minscore num minscorebylength toppercent num winscore num minsupport num Import BLAST and reads file and create a new MEGAN file open meganfile name Open the named MEGAN file save meganfile name summary bool Save summary or comparison to a file export data reads blast file name taxid num Export all reads or matches If taxid 0 only those assigned to the givent taxon export data CSV file name format readid_taxonname taxonname_count taxonname_readid readid_taxonid taxonid_count taxonid_readid Export reads or counts in CSV comma separated values format Setting thresholds and options set minscore num Set the minimum bit score set minscorebylength num Set the minimum bit score divided by read length set toppercent num Set the percentage win against top score set winscore num Set the win score set minsupport num Set the minimum number of reads that must support a taxon enable labels selected Enable all selected taxa enable all Enable all taxa disable labels selected Disable all selected taxa
11. ed eee we amp 4 10 Window Memi soso we A A A A ee ee one oa 2 RAE SS Thee Tool BOP a a ae feeds E 158 Popup Menus 0 6 2 ebb dee Pe ee a eee ead PSE wed a eee 158 Wheel Mouse and Special Keys cc ee ewe ee hE wae ee ee a 16 Format Dialog 17 Message Window 18 Parameters Dialog 19 Compare Dialog 20 Extractor Dialog 21 Export Image Dialog 22 About Window 23 File Formats 23 1 The MEGAN Text File Format 23 2 Full Summary and Comparison MEGAN Files lt q ca sasora e e 17 18 18 19 20 20 21 22 22 23 23 23 23 24 24 24 24 25 25 25 26 23 3 Required Syntax of BLAST Files oss haa eee RR HE eH 28 234 Required Format of Read Files a ce caca 422444 24 odd dd ddaa aeea 28 Dou Graphics Forma e o a a Re Ga we a PR ep eR Oa RS SE a Goa A ae E Be ww 28 Bo A Pee oboe a A a RR eae SE Se ae ee OL ES Bee GG a 31 23 Tree and Map Format oo rara aaa 32 24 Command Line Options and Mode 32 25 Examples 35 26 Using More Memory 35 27 Acknowledgments 35 References 35 Index 37 1 Introduction Disclaimer This software is provided AS IS without warranty of any kind This is develop mental code and we make no pretension as to it being bug free and totally reliable Use at your own risk We will accept no liability for any damages incurred through the use of this software Use of the MEGAN is free however the program is not open source Type setting conventions In this manual we
12. file 22 About Window The About Window is opened using the Window About item It reports the version of the program 23 File Formats MEGAN uses its own file formats to store the data describing the result of a sequence comparison computation between a file of DNA reads and a database of reference sequences such as computed by BLASTX BLASTP or BLASTN 1 Files ending in rma are in a compressed binary format called RMA read match archive which is a new open format that we will describe in a separate document MEGAN 1 used a text format files ending on megan or meg which are now dep recated and will not be supported by futher versions of the program By convention we use the suffix megan for MEGAN text files and rma for binary read match archive files A RMA file is generated using the File Import BLAST menu item from a BLAST file and a read file A RMA file contains all reads and all significant BLAST matches by default up to 100 matches per read in a compressed format which we call read match archive RMA format The size of such a file is around 10 20 of the size of the original input files and is thus usually smaller than the file that one obtains by simply compressing the BLAST file The file is indexed and thus provides MEGAN with fast access to data stored in it The reads and matches can be extracted from the file and so the MEGAN file provides a means of keeping all reads BLAST matches and analysis in one documen
13. i e don t use them when placing reads set ignore_duplicate_matches bool ignore duplicate matches in data set ignore_nohits bool ignore reads with no hits in data set useparsetext bool option for import command parse text embedded in BLAST file to identify taxa set usesynonyms bool open lookupdir dir option for import command use synonyms imported from the given directory set useaccessionlookup bool open lookupdir dir option for import command use accession number lookup tables imported from the given directory Comparison of multiple datasets compare mode absolute relative pid num pid num Show comparison of different datasets in new window GUI mode compare mode absolute relative meganfile name meganfile name Compute comparison of different datasets command line mode Listing information list summary all Summarize assignment of reads to all nodes list summary selected Summarize assignment of reads to selected nodes list summary assigned Summarize assignments 33 list COGs all Summarize COGs for all nodes list COGs all Summarize COGs for all selected nodes list reads2hits Lists all reads and hits use only for small datasets list key name label name List for each key how many reads hit the key list strong threshold num List the number of strong nodes for given threshold list disabled List all disabled taxa Collapsing and uncollapsing nodes collapse all Collapse all nodes collapse sel
14. is around 20 of the size of the original input files and is thus usually smaller than the file that one obtains by simply compressing the BLAST file MEGAN also provides the option of saving an analysis as a summary only A summary contains only information on how many reads were assigned to each taxon The analysis can not be changed or queried The corresponding file is very small MEGAN supports import of data from other programs in a comma separated format from a CSV file 6 The NCBI Taxonomy The NCBI taxonomy provides unique names and IDs for over 350 000 taxa including approx imately 25 000 prokaryotes 84 000 animals 65 000 plants and 17 000 viruses The individual species are hierarchically grouped into clades at the levels of Superkingdom Kingdom Phylum Class Order Family Genus and Species and some unofficial clades in between At startup MEGAN automatically loads a copy of the complete NCBI and then displays the tax onomy as a rooted tree The taxonomy is stored in an NCBI tree file and an NCBI mapping file which are supplied with the program 7 The NCBI NR and NCBI NT Databases The NCBI NR non redundant protein sequence database is available from the NCBI website It contains entries from GenPept Swissprot PIR PDF PDB and RefSeq It is non redundant in the sense that identical sequences are merged into a single entry The NCBI NT nucleotide sequence database is available from the NCBI website It co
15. selected hits are given the status to be ignored Such hits are ignored by all algorithms and are not used to decide where to place a given read All hits that have been marked in this way are shown in red This item is also available via right clicking in the window The Options Use Hit item is used to remove the to be ignored status from all selected hits This item is also available via right clicking in the window The Options Use All Hits item is used to remove the to be ignored status from all hits The Options Apply Ignore Use Changes item is used to rerun the taxonomical analysis of the dataset taking the change of the to be ignored status of hits into account Find Window Find window can be opened using the Edit Find item It s purpose is to find taxa or reads Enter a query specifying a name or ID of a taxon in the top text region Use the following check boxes to parameterize the search If the Whole words only item is selected then only taxa or reads matching the complete query string will be returned If the Case sensitive item is selected then the case of letters is distinguished in com parisons 19 e Ifthe Regular Expression item is selected then the query is interpreted as a Java regular expression Press the Close Find First or Find Next buttons to close the dialog or find the first or next occurrence of the query respectively Press the Find All button to find all occur
16. the MEGAN website 26 Using More Memory To run MEGAN with more than 2GB under MacOS X on an intel Mac edit the file Applications MEGAN MEGAN app Contents Info plist as follows Find the lines lt key gt VMOptions lt key gt lt string gt server Xmx1600M lt string gt lt I4J_INSERT_VMOPTIONS gt and replace then by lt key gt VMOptions lt key gt lt string gt server d64 Xmx4000M lt string gt lt I4J_INSERT_VMOPTIONS gt to run using 4GB for example To run MEGAN with more than 2GB on a 64 bit unix linux system open the file Applications megan MEGAN in a text editor Find the current memory specification e g Xmx1600M and replace it by the following d64 Xmx4G to run with 4 gigabytes of mem ory say Note that the flag d64 is necessary to specify 64 Bit Java 27 Acknowledgments This product includes software developed by the Apache Software Foundation http www apache org namely the batik library for generating image files It also contains JFreeChart to construct charts BrowserLauncher2 for opening browser windows Text for generating pdf files and MRJAdapter a Java package used to help construct user interfaces for the Apple Macintosh Licenses can be found in the installation directory References 1 S F Altschul T L Madden A A Schaffer J Zhang Z Zhang W Miller and D J Lipman Gapped BLAST and PSI BLAST a new generation of protein database search programs Nu cleic A
17. use e g Edit Find to indicate the Find menu item in the Edit menu How to cite If you publish results obtained in part by using MEGAN then we require that you acknowledge this by citing the program as follows e D H Huson A Auch Ji Qi and S C Schuster MEGAN analysis of metagenome data Genome Research 17 377 386 2007 software freely available for academic purposes from www ab informatik uni tuebingen de software megan The term metagenomics has been defined as The study of DNA from uncultured organisms Jo Handelsman and an approximately 99 of all microbes are believed to be unculturable A genome is the entire genetic information of one organism whereas a metagenome is the entire genetic information of an ensemble of organisms Metagenome projects can be as complex as large scale vertebrate projects in terms of sequencing assembly and analysis The aim of MEGAN is to provide a tool for studying the taxonomical content of a set of DNA reads typically collected in a metagenomics project In a preprocessing step a sequence comparison of all reads with a suitable database of reference DNA or protein sequences must be performed to produce an input file for the program At start up MEGAN first reads in the current NCBI taxonomy consisting of around 460 000 taxa A first application of the program is that it facilitates interactive exploration of the NCBI taxonomy However the main application of the program is to parse
18. In this section we give an overview over the main design goals and features of this program Basic knowledge of the underlying design of the program should make it easier to use the program MEGAN is written in the programming language Java The advantages of this is that we can provide versions that run under the Linux MacOS Windows and Unix operating systems Typically after generating a RMA file read match archive from a BLAST file the user will then interact with the program using the Find window to determine the presence of key species collapsing or un collapsing nodes to produce summary statistics and using the Inspector window to look at the details of the matches that are the basis of the assignment of reads to taxa The assignment of reads to taxa is computed using the LCA assignment algorithm see 3 for details The program is designed to operate in two different modes in a GUI mode the program provides a GUI for the user to interact with the program In command line mode the program reads commands from a file or from standard input and writes output to files or to standard output 5 Importing Reading and Writing Files To open an existing RMA file or MEGAN file select the File Open menu item and then browse to the desired file Alternatively if the file was recently opened by the program then it may be contained in the File Open Recent submenu New input to the program is usually provided as a BLAST file obtained fro
19. Show Legend item determines whether to show or hide the data sets legend in the main window By default this is off for single datasets and on for comparisons The Edit Preferences Edit Comparison Colors item can be used to change the colors used in a comparison of datasets Select Menu Select menu contains items for selecting different sets of nodes in the taxonomy The Select gt A11 Nodes item is used to select all nodes The Select None item is used to deselect all nodes 11 11 4 The The Select From Previous Window item applies the selection in window previously on top to the window currently on top This feature is useful for comparing the contents of different windows The Select gt A11 Leaves item is used to select all leaves The Select gt A11 Internal Nodes item is used to select all internal nodes The Select gt A11 Intermediate Nodes item is used to select all intermediate nodes that is nodes with exactly one in edge and one out edge The Select gt Subtree item is used to select all nodes below any currently selected node The Select Invert item is used to invert the current node selection The Select gt Level item opens a sub menu that can be used to selecte taxa by their taxonomical level such as Kingdom Phyla Class Order Family etc Layout Menu Layout menu contains items that control aspects of the visualization of the tree The Layout Expand Contract item provides a submenu for expandin
20. So any preferences set using the interactive version of the program will 32 also apply to the command line version of the program It this is not desired then please use the p option to supply a different properties file Another important thing to note is that the command parser operates in a line by line fashion When processing commands in a given line the parser makes note of required updates to the taxonomy and data structures These updates are not executed until all commands in the current input line have been processed For example if you want to open and MEGAN file and then to save a picture of the taxonomical analysis in a PDF file then the two commands should be entered on separate lines because otherwise the taxonomy will be drawn before the data from the MEGAN file has been processed Here is an example of the correct way to produce a picture of a dataset open meganfile myfile rma exportgraphics format PDF file myfile pdf Alternatively the update command can be used to explicitly force MEGAN to update all data structures e g open meganfile myfile rma update exportgraphics format PDF file myfile pdf As described below the update command takes a number of different parameters that can be used to determine exactly what type of update is required All commands supplied using the x command line option are parsed as if they were contained in one line So here the update command must be used to ensure that commands are
21. User Manual for MEGAN V3 8 Daniel H Huson and Stephan C Schuster with contributions from Alexander F Auch Daniel C Richter Suparna Mitra and Qi Ji February 4 2010 Contents Contents 1 1 Introduction 3 2 Getting Started 5 3 Obtaining and Installing the Program 5 4 Program Overview 6 5 Importing Reading and Writing Files 6 6 The NCBI Taxonomy 7 7 The NCBI NR and NCBI NT Databases 7 8 Identification of COGs 7 9 Assigning Reads to Taxa 8 10 Assigning Reads to Gene Ontology Terms 8 11 Main Window 9 ILL Fie ieee o a BA A ee AEE EE A e oh ee BS LA UE MA ec ch a Ee oe RS A ee Be Bee A A 11 TILA peket Me o og ce kk Eo ee ass ras de 11 lA LASA ME ook a DE Se he eee ee Re A A ee ee oe 8 12 11 5 Options Memi os c c ee oe a ee a eR A ee eee Ge 13 VLG Tre Memeo quu anane a eee a a RR A A eee ew 4 14 Ia ARTE Ra RE DSR RRS ERE TLS MEGAN Ment sec 2 Baa Ga be ee eee Pa eee HORE ER Rae ee HY VES TA A see Se E a SS Se ee Se Bg oe a ee Be UM IWPSpup Menus o ee ii ee ew AAA AAA A oe a 11 11Wheel Mouse and Special Keys e oo ir a a 12 Import Dialog 13 Inspector Window 131 Inspector Menus a ee a SE E AR a a a 14 Find Window 15 GOAnalyzer Window 15 1 Exploring the Read Assignments lt o o ee 15 2 Bile Menu lt s eces eee ee eA ee eed eee be eee ee el deed Soe a bs a A eae aes Se a Bek Be eee ae casi oe eee Sy SS 14d Options Moda on OE oa ees ee Gb bade EEO eee ee eee Ow a loo View Memi ia A a ee eee et RA A
22. abase sequence must contain a NCBI taxon name If names are not contained in the comment lines then the accession lookup support must be used 30 23 6 CSV Files MEGAN supports importing data from other programs in a comma separated format froma CSV file using the File Import CSV menu item The input file must be a text file in which either all lines each contain two strings that are separated by a comma or all lines each contain three strings separated by commas Importing read assignments If each line of the CSV file contains two strings separated by a comma then the first string will be intepreted as a taxon name or taxon id and the second string will be intepreted as an integer specifying the number of reads assigned to the named taxon MEGAN will assume that this is the result of some analysis and thus will produce a summary file from it and will simply display it on the NCBI taxonomy with no further analysis For example assume that you have done a metagenome analysis using some other method and have obtained the following result e Gammaproteobacteria 55 reads e Mollicutes 400 reads e Escherichia coli K12 42 reads e Unknown 100 reads To import this data into MEGAN so as to visualize the taxonomical assignments produce the following CSV file Gammaproteobacteria 55 Mollicutes 400 Escherichia coli K12 42 Unassigned 100 MEGAN will draw a tree with four nodes one for each of the named taxa Importing
23. alignments were found In this case use the Options Set Number of Reads menu item to set the total number of reads in the analysis Clicking on a node will cause the program to display the exact number of hits of any given node and the number of hits in the subtree rooted at the node Right clicking on a node will show a popup menu and selecting the first item there Inspect will open the Inspector window which is used to explore the hits associated with any given taxon A node is selected by clicking on it Double clciking on a node will select the node and the whole subtree below it Double clicking on the label of a node will open the node in the Inspector window Example files are provided with the program They are contained in the examples subdirectory of the installation directory The precise location of the installation directory depends upon your operating system 3 Obtaining and Installing the Program MEGAN is written in Java and requires a Java runtime environment version 1 5 or newer freely available from www java org MEGAN is installed using an installer program that is freely available from www ab informatik uni tuebingen de software megan There are four different installers targeting different operating systems e MEGAN windows_3 8 exe provides an installer for Windows e MEGAN macos_3 8 dmg provides an installer for MacOS e MEGAN unix 3 8 sh provides a shell installer for Linux and Unix 4 Program Overview
24. axa H and assign the read r to the taxon represented by v We call this the LCA assignment algorithm LCA lowest common ancestor In this approach every read is assigned to some taxon If the read aligns very specifically only to a single taxon then it is assigned to that taxon The less specifically a read hits taxa the higher up in the taxonomy it is placed Reads that hit ubiquitously may even be assigned to the root node of the NCBI taxonomy The program provides a threshold for the bit score of hits Any hit that falls below the threshold is discarded Secondly a threshold can be set to discard any hit whose score falls below a given percentage of the best hit Finally a third threshold can be used to report only taxa that are hit by a minimal number of reads By default the program requires at least two reads to hit a taxon before the taxon is deemed present Taxa in the NCBI taxonomy can be excluded from this analysis For example taxa listed under root unclassified sequences metagenomes may give rise to matches that force the algorithm to place reads on the root node of the taxonomy This feature is controlled by Options Taxon Disabling menu At present the set of disabled taxa is saved as a program property and not as part of the Megan document 10 Assigning Reads to Gene Ontology Terms Besides the taxonomical analysis MEGAN provides functionality to obtain information about the functional content of a metagenomi
25. ay exclude some important matches By default the 100 highest scoring matches per read are save If the Save As Summary Only check box is selected then the data will be saved in a small summary file rather than a full RMA file A summary contains only information on how many read where assigned to each taxon The analysis can not be changed or queried The corresponding file is very small The fourh tabbed pane titled the LCA Parameters pane contains all items of the Parameters dialog which allows one to set the parameters used by the LCA algorithm Because re computation 17 of an analysis can take quite long on a very large dataset it is recommended to set these values at this stage The last tabbed pane titled the Advanced Options pane controls how MEGAN attempts to identify the taxon associated with a given BLAST hit By default MEGAN looks for the name of a taxon in the header line of the subject sequence which is the fastest option The Parse taxon names checkbox specifies that the program first attempts to obtain the taxon name from the BLAST hit header lines The Load Accession Lookup opens a menu that can be used to load the accession lookup directory This directory contains a number of binary format files used by MEGAN to map accession numbers to taxon ids and taxon names This directory is very large and thus not part of the MEGAN distribution It can be downloaded from http www ab informatik uni tuebingen de software megan The
26. c data set Therefore a module named GOAnalyzer assigns read matches derived from a BLASTX comparison against the NCBI NR database to terms of the Gene Ontology GO see http www geneontology org GO provides three sets of structured vocabularies that describe biological processes molecular functions and cellular components Each of these three ontologies is represented by a directed acyclic graph DAG that contains uniquely defined GO terms as nodes and the relationships among them as edges GO is hierarchically structured i e GO terms can be parent of child terms e g taxis is a child term of behavior and child terms may have more than one parent term The GOAnalyzer uses the header information of BLAST hits and a pre computed mapping file to assign environmental reads to GO terms The mapping is based on RefSeq identifiers http www ncbi nlm nih gov RefSeq and uses the associations provided in ftp ftp pir georgetown edu databases idmapping idmapping tb gz To reduce complexity we use a variant of the LCA algorithm to modify the mapping such that each RefSeq identifier maps to at most three GO terms one for each of the three ontologies When blasting reads against a database most reads that have hits usually map to multiple entries These often correspond to different RefSeq identifiers and thus different GO terms By applying the LCA algorithm each read is mapped to at most one GO term in each of the three ontologies
27. cids Res 25 3389 3402 1997 2 D A Benson I Karsch Mizrachi D J Lipman J Ostell and D L Wheeler Genbank Nucleic Acids Res 1 33 Database issue D34 38 2005 35 3 D H Huson A F Auch J Qi and S C Schuster MEGAN analysis of metagenomic data Genome Res 17 3 377 386 March 2007 4 Hendrik N Poinar Carsten Schwarz Ji Qi Beth Shapiro Ross D E Macphee Bernard Buigues Alexei Tikhonov Daniel H Huson Lynn P Tomsho Alexander Auch Markus Rampp Webb Miller and Stephan C Schuster Metagenomics to paleogenomics large scale sequencing of mammoth dna Science 311 5759 392 394 Jan 2006 5 S Schwartz W J Kent A Smit Z Zhang R Baertsch R C Hardison D Haussler and W Miller Human mouse alignments with BLASTZ Genome Res 13 103 107 2003 36 Index m 0 28 m 7 28 m 8 28 meg 26 megan 26 rma 26 About 15 16 26 accession lookup directory 18 Advanced Options pane 18 Algorithm 27 All Intermediate Nodes 12 All Internal Nodes 12 All Leaves 12 All Nodes 11 Antialiased Painting 22 Apply Ignore Use Changes 19 Assignments 10 Backward 20 batik 35 Blast 10 BLAST file 28 BLAST hits 20 BLASTN file 28 BLASTP file 28 BLASTX file 28 BMP 28 BrowserLauncher2 35 Case sensitive 19 Change LCA Parameters 13 24 Chart COGs 15 Chart GO 23 Chart Microbial Attributes 15 Chart Taxa 15 Class 12 14 Clear 19 Close 11 18 20 21
28. des at Taxonomical Level 14 Tree Labels Off 15 Tree Labels On 15 Tree Show Intermediate Labels 15 Tree Show Number Of Reads Assigned 14 Tree Show Number Of Reads Summarized 15 Tree Show Taxon IDs 14 Tree Show Taxon Names 14 Tree Uncollapse 5 14 Tree Uncollapse Subtree 5 14 Type setting conventions 3 unassigned 24 ncollapse 5 14 16 ncollapse Subtree 5 14 16 nix 6 pdate 33 se absolute counts 25 se Accession Lookup 18 se All Hits 19 se Hit 19 se Synonyms 18 coad cacade version COGs 11 version NCBI microbial attributes 11 version NCBI taxonomy 11 vertical zoom 17 View Fit Content 23 View Full View 23 View Generic GO Slim 23 View gt GOA and Proteome GO Slim 23 View Plant GO Slim 23 View Prokaryotic Subset 23 View Show All 3 ontologies 23 View Yeast GO Slim 23 Website 15 Whole words only 19 Win Score 25 Window 15 Window About 15 16 26 Window Chart COGs 15 Window Chart GO 23 Window Chart Microbial Attributes 15 Window Chart Taxa 15 Window Command syntax 15 Window Enter a command 15 Window How to cite 15 Window Inspector Window 15 18 Window Message Window 15 24 Window Microbial Attributes Window 15 Window Register 15 Window Website 15 Windows 5 6 Wizard pane 17 Yeast GO Slim 23 Zoom to Selection 13 42
29. e Collapse can be used to collapse the subtree below a selected node thus summarizing the subtree by the node The Tree Collapse Nodes at Level prompts the user for the input of a level and then collapses all nodes whose distance number of edges to the root of the tree equals the given level By default if no data is given the program displays the full NCBI taxonomy collapsed at level 2 The Tree Collapse Nodes at Taxonomical Level provides a submenu which allows the user to collapse nodes at the level of Kingdom Phyla Class Order Family etc The Tree Uncollapse item un collapses a selected collapsed node by displaying all the children of the node The Tree gt Uncollapse Subtree item un collapses the whole subtree below a selection of nodes Ifthe Tree Show Taxon Names item is selected nodes are labeled by NCBI taxon names If the Tree Show Taxon IDs item is selected nodes are labeled by NCBI IDs Ifthe Tree Show Number Of Reads Assigned item is selected nodes are labeled by the number of reads assigned to the corresponding taxa 14 11 7 The If the Tree Show Number Of Reads Summarized item is selected nodes are labeled by the number of reads assigned to the corresponding taxa or to any that contained in the subtree The Tree Labels On item sets the label of selected nodes to visible The Tree Labels Off item sets the label of selected nodes to invisible If the Tree Show Interm
30. e Layout Draw Circles item ensures that nodes are drawn as circles Please note that the size of circles is scaled logarithmically The Layout Draw Pie Charts item ensures that nodes are drawn as pie charts Please note that the size of each pie chart is scaled logarithmically to indicate the total number of reads assigned to the node but the proportions of the pie assigned to different datasets is scaled linearly by the number of reads The Layout Draw Heat Maps item ensures that nodes are drawn as heat maps Please note that colors are scaled logarithmically The Layout Draw Heat Maps 2 shows a pairwise comparison of two or more datasets as a heat map for each node Please note that colors are scaled logarithmically The Layout Draw Meters item ensures that nodes are drawn as meters Please note that the meters are scaled logarithmically The Layout Draw Leaves Only item ensures that only leaves are drawn The Layout Highlight Differences item turns on a simple statistical test that high lights significantly different nodes in a comparison of two datasets 11 5 Options Menu The Options menu contains the following items The Options Change LCA Parameters item opens the Parameters dialog that allows one to change the parameters used by the LCA algorithm and to then rerun the analysis The Options Taxon Disabling sub menu contains menu items for disabling taxa or enabling taxa Disabled taxa are ignored by the algo
31. e length of the read in the format length length or as length length Another important feature is that the comment line of the database sequence must contain a NCBI taxon name If names are not contained in the comment lines then the accession lookup support must be used Finally the Gaps statement is optimal 29 BLASTN text text followed by 0 or more blocks of the following type Query query id text length length text or Query query id text length length text text followed by 0 or more blocks of the following type gt text NCBI taron name text line breaks ok Score score bits bits Expect e value Identities text percent identities Gaps text percent gaps h Strand strand strand followed by O or more blocks of the following type Query text text Sbjct text Figure 2 The required structure of a BLASTN file Labels shown as label are tokens that must occur verbatim in the file Labels shown as label are values that are read into the program The first word in the file must be BLASTN The header line starting with Query which is taken from the Fasta header of the query sequence a read must start with a one word unique identifier for the read and must also contain a statement containing the length of the read in the format length length Another important feature is that the comment line of the dat
32. e representing an alignment of the given read to a sequence associated with some taxon Finally double clicking on a read hit node will display the actual BLAST alignment provided to deduce the relationship 13 1 Inspector Menus The Inspector window has three menus The File menu contains the following items e The File Save As saves the currently displayed data to a file not implemented e The File Print prints the currently displayed data not implemented e The File Close item closes the Inspector window The Edit menu contains the following items 18 The 14 The The Edit Select All item is used to select the whole text The Edit gt Cut item is used to cut text The Edit gt Copy item is used to copy text The Edit gt Paste item is used to paste text The Edit gt Clear item is used to clear all displayed data Options menu contains the following items The Options Show Taxon item prompts the user for a taxon name or ID and then adds the named taxon to the list of displayed data if at least one read has been assigned to the taxon The Options Show Read item prompts the user for a read ID and then adds the named read to the list of displayed data If the Options Collapse item is clicked the subnodes of the highlighted entry are collapsed Ifthe Options Expand item is clicked the subnodes of the highlighted entry are displayed If the Options Ignore Hit item is clicked then all currently
33. e used to to save comparison files or summary files A RMA file is kept sycronized with the program and thus need not be saved e The File Import BLAST item is used to import new data into MEGAN The user is presented with a Import wizard panel which can be used to specify the BLAST file and reads file to import and the name of the new RMA file to create The Import wizard contains additional tabbed panes for advanced users to set additional options reads file and BLAST file back out of the project The File Export Assignments menu is used to export a summary of the read assignments in comma separated values CSV format There are a number of possible listings to export x Select read id taxon name to list read identifiers and the names of the taxa that they have been assigned to Select read id taron id to list read identifiers and the ids of the taxa that they have been assigned to Select taxon id count s to list taxon ids and the number of reads that have been assigned to each taxon If applied to a comparison file the first line of the output will contain all file names and then the subsequent lines will contain the numbers for each file The numbers return reflect the number of reads assigned to a node unless the node is a leaf of the currently visible taxonomy in which case the number of reads summarized by the node is returned x Select taxon name count s to obtain the same output as for the previous ite
34. ead hit node 18 read node 18 read id taxon id 10 read id taxon name 10 read match archive 26 Reads 10 20 25 reads file 28 Register 15 Regular Expression 20 RMA 26 RMA file 26 Save As 9 18 Save As Summary Only 17 Save visible image 25 Save whole image 25 40 Scale Nodes By Assigned 12 Scale Nodes By Summarized 12 Select 11 Select All 19 Select Subgraph 22 23 Select All Intermediate Nodes 12 Select All Internal Nodes 12 Select All Leaves 12 Select All Nodes 11 Select From Previous Window 12 Select Invert 12 Select Level 12 Select None 11 Select Subtree 12 Set Label Font Size 22 Set Max Node Radius 12 Set Number of Reads 5 14 Show All 3 ontologies 23 Show Colored Read Assignment Table 22 Show GO Term in List 22 24 Show Intermediate Labels 15 Show Legend 11 Show Node Labels 22 Show Number Of Reads Assigned 14 Show Number Of Reads Summarized 15 Show Read 19 Show Taxon 18 19 Show Taxon IDs 14 Show Taxon Names 14 Source 27 Subtree 12 Summary 10 27 summary 7 28 summary file 10 17 SVG 28 Synchronize GO Term Selection 22 synonyms file 18 Taxon Disabling 8 13 taxon node 18 taxon id count s 10 taxon id read id s 10 taxon name count s 10 taxon name read id s 10 Tools 11 Top Percentage 25 TotalReads 27 Tree 14 Tree Collapse 14 Tree Collapse Nodes at Level 14 Tree Collapse No
35. ection Collapse all selected nodes collapse level number Collapse taxonomy at the given numerical level collapse level name Collapse taxonomy at the named taxonomical level collapse taxa t1 t2 Collapse all named taxa uncollapse all Uncollapse all nodes uncollapse selection Uncollapse all selected nodes uncollapse subtrees Uncollapse whole subtree for selected nodes uncollapse taxa t1 t2 Uncollapse all named taxa show tree full Show the full taxonomy show tree induced Show the induced taxonomy Visualization set nodedrawer name Sets the node drawer circle piechart heatmap heatmap2 or meters set drawleavesonly boolean Draw leaves only set fontsize number Set font size set autolayoutlabels boolean Set auto layout of labels on off set margin left num right num top num bottom num Set the margin around the tree show labels selected Display labels for all selected nodes hide labels selected Hide labels for all selected nodes hide labels intermediate Hide labels for all intermediate nodes nodelabels names bool ids bool assigned bool summarized bool Set what to label nodes by nodesize scaleby summary assigned Set whether to scale nodes by summary or assigned reads set highlightdifferences bool In a comparison of two datasets turn difference hightlighting on or off Scaling expand direction horizontal Expand image horizontally contract direction horizontal Contract image horizontally expand direction ver
36. ediate Labels item is selected the labels of all intermediate nodes of degree two in the induced taxonomy are shown By default this is turned off Window Menu Window menu contains the following items The Window gt About item shows information about the version of MEGAN When the program is run under MacOS this menu item appears in the MEGAN menu The Window How to cite item gives instructions on how to cite the program The Window gt Website item opens the programs website in a browser The Window Register item allows the user to register their copy of the program using a key obtained from the program website The Window Message Window item opens the Message window and brings it to the front The Window Inspector Window item opens the Inspector window that can be used to inspect the alignments that are the basis of the assignment of reads to taxa The Window Microbial Attributes Window items opens a new window showing var ious physiological features associated with a each read assigned microbial organism The classification is adapted from the NCBI microbial attributes table The Window Chart Taxa item opens a window that provides different types of charts summarizing taxon assignments The Window Chart COGs item opens a window that provides different types of charts summarizing COG assignments The Window Chart Microbial Attributes item opens a window that provides different types of charts summari
37. ee values Full Dataset Summary or Comparison Ina full dataset file each line is assumed to contain a description of one read or read hit Ina summary file each line is assumed to contain the a taxon and the number of reads that have been assigned to it In a comparison file each line is assume to contain a taon and the number of reads that have been assigned to it for two or more datasets which are specified further in the CFormat line Future versions of MEGAN might not support the full dataset format 23 3 Required Syntax of BLAST Files MEGAN imports data froma BLAST file MEGAN can parse BLAST files in standard or XML format obtained using the BLAST output option m O or m 7 respectively MEGAN can also parse tabular format BLAST output option m 8 however this format is generally not suitable for MEGAN because it doesn t contain the information required to determine the taxon or COG associated with a matched sequence MEGAN can read gzipped BLAST files For human readable format any BLASTX file or BLASTP file is expected to adhere to the format shown in Figure 1 Any BLASTN file is expected to adhere to the format shown in Figure 2 23 4 Required Format of Read Files Reads from sequencing are assume to be provided in multi FastA format in a reads file The first word of a FastA header is assumed to be the read id The remaining text of the FastA header must contain the length of the read either as length number or as lengt
38. elected edges 15 9 Wheel Mouse and Special Keys Use of a wheel mouse is recommended for zooming of the GOAnalyzer window To scroll the graph either press and drag the mouse using the right mouse button or use the arrow keys 16 Format Dialog The Format dialog is opened using the Edit gt Format item This is used to change the font color size and line width of all selected nodes and edges Also it can be used to turn labels on and off 17 Message Window The Message window is opened using the Window Message Window item The program writes all messages to this window The window contains the usual File and Edit menu items 18 Parameters Dialog The Parameters dialog is used to control the parameters of the LCA assignment algorithm It can be invoked by selecting Options Change LCA Parameters The dialog options are e The Min Support item can be used to set a threshold for the minimum support that a taxon requires that is the number of reads that must be assigned to it so that it appears in the result Any read that is assigned to a taxon that does not have the required support is counted as unassigned By default the minimum number of reads required for a taxon to appear in the result is 5 e The Min Score item can be used to set a minimum threshold for the bit score of hits Any hit in the input data set that scores less than the given threshold is ignored e The Min Score Length item can be used to set a minimum th
39. g or contracting the picture of the tree to a certain degree The Layout Expand Contract Expand Horizontal item expands the picture of the tree horizontally The Layout Expand Contract Contract Horizontal item contracts the picture of the tree horizontally The Layout Expand Contract Expand Vertical expands the picture of the tree vertically The Layout Expand Contract Contract Vertical contracts the picture of the tree vertically The Layout Font Size item is used to set the font size of all labels on the tree If the Layout Layout Labels item is checked then the program will attempt to layout node labels in a none overlapping fashion If the Layout Scale Nodes By Assigned item is selected then the size of every node is scaled by the number of reads assigned to the corresponding taxon Ifthe Layout Scale Nodes By Summarized item is selected then the size of every node is scaled by the number of reads assigned to the corresponding taxon or assigned to any taxon below the node The Layout gt Set Max Node Radius allows the user to specify the maximum size in pixels a node can obtain 12 The Layout Zoom to Selection item is used to zoom to all selected nodes and edges in the tree The Layout Fully Contract item is used to contract the picture of the tree to its smallest size The Layout Fully Expand item is used to expand the picture of the tree to its largest size Th
40. h length 23 5 Graphics Formats The following graphics formats are supported e BMP Bitmap e EPS Encapsulated PostScript vector format e GIF Graphics Interchange Format e JPEG Joint Photographic Experts Group e PDF Portable Document Format vector format e PNG Portable Network Graphics e SVG Scalable Vector Graphics vector format 28 BLASTX text text followed by 0 or more blocks of the following type Query query id text length length text or Query query id text length length text text followed by 0 or more blocks of the following type gt text NCBL taxon name text line breaks ok Score score bits bits Expect e value Identities text percent identities Positives text percent positives Gaps text percent gaps h Frame frame followed by 0 or more blocks of the following type Query text text Sbjct text Figure 1 The required structure of a BLASTX file Labels shown as label are tokens that must occur verbatim in the file Labels shown as label are values that are read into the program The first word in the file must be BLASTX The header line starting with Query which is taken from the Fasta header of the query sequence a read must start with a one word unique identifier for the read and must also contain a statement containing th
41. hat provide short cuts to some of the menu items associated with the window These are the Zoom in and out button View Fit Content Edit Find Options Inspect GO Options gt Chart GO Options Extract Reads By GOs Draw Nodes As Rectangles Draw Nodes As Pie Charts Draw Nodes As Heatmaps Draw Nodes As Pairwise Comparison Heatmaps Draw Nodes As Meters and a drop down list providing quick access to the View gt Full View or the GO slims 15 8 Popup Menus The GOAnalyzer window provides two different popup menus that are activated by right clicking on a node or an edge If are using a single button mouse under MacOS then please control click to access these menus The popup menu that is opened when a node is right clicked on has the following items e The Copy GO ID s copies the GO ID s of all selected nodes e The Copy GO Names copies the GO term names of all selected nodes e The Select Subgraph item is used to select all child nodes of currently selected nodes 23 e The Highlight Paths of Selected Nodes item is used to highlight all paths of the currently selected nodes e The Options Show GO Term in List item is used select and focus the GO terms in the list of the corresponding selected nodes in the graph view The popup menu that is opened when an edge is right clicked on has the following items e The Options Highlight Incident Nodes of Selected Edges item is used select all incident nodes of currently s
42. ifile name Extract taxonomy from NCBI dump file Charting chart taxa Chart taxonomical analysis chart go Chart all occurrences of GO terms chart cogs summy bool Chart all occurrences of COGs chart attributes Chart all microbial attributes Additional computations extract outdir outDir outfile outFileNameTemplate summarized bool taxa taxon names cogs COG ids gos G0 ids Extract all reads that are assigned to any named taxon COG ids or GO ids When extracting by taxa report all reads on or below taxon if summarized true In outFileNameTemplate every occurrence of t is replaced by the corresponding taxon name subsample percent num Randomly select a subset of reads Other exportgraphics format EPS PNG GIF JPG SVG PDF replace bool textasshapes bool title title file filename Export a picture of the current tree recompute minsupport num minscore num minscorebylength num toppercent num winscore num Rerun the LCA analsyis with different parameters dump file name Dump the complete contents of an RMA file to a human readable file update reprocess bool reset bool reinduce bool Update the computation set window width num height num x num y num Size and location of main window show vint bool Show version string in title of windows help List this help about List information about MEGAN version List version info 34 quit Quit the program 25 Examples Example files can be downloaded from
43. ighlight all paths of the currently selected nodes e The Options Highlight Incident Nodes of Selected Edges item is used to select all incident nodes of currently selected edges e The Options Show GO Term in List item is used select and focus the GO terms in the list of the corresponding selected nodes in the graph view e The Options Inspect GO item is used to display the currently selected GO terms in the Inspector window e The Options Extract Reads By GOs item is used to extract all read sequences assigned to the selected GO terms 22 15 5 View Menu e The View Show All 3 ontologies item is used to display all three ontologies whole GO graph e The View gt Fit Content item is used to fit the view to the window size e The View Generic GO Slim item is used switch to the Generic GO Slim e The View GOA and Proteome GO Slim item is used to switch to the GOA and Proteome GO Slim e The View Plant GO Slim item is used to switch to the Plant GO Slim e The View Prokaryotic Subset item to switch to the Prokaryotic Subset of GO terms e The View Yeast GO Slim item is used to switch to the yeast GO Slim e The View Full View item is used display the full Gene Ontology instead of slim versions 15 6 Window Menu e The Window Chart GO item is used to open a chart window displaying the selected GO terms as bar or pie chart 15 7 Tool Bar The GOAnalyzer window provides a tool bar containing buttons t
44. m but using taxon names instead of ids Select taxon id read id s to list taxon ids and the ids of all reads assigned to each taxon Select taron name read id s to obtain the same output as for the previous item but using taxon names instead of ids The File Export Reads menu item is used to export all reads from the project If any nodes are selected then only the reads assigned to those nodes are exported The File Export Blast menu item is used to extract all BLAST matches from the project If any nodes are selected then only the BLAST matches of reads assigned to those nodes are exported The File Export Summary menu item can be used to generate a summary file from a given project A summary contains only information on how many read where assigned to each taxon The analysis can not be changed or queried The corresponding file is very small The File Export Image item opens the Export Image dialog which is used to save the current tree in a number of different graphics formats see Section 23 5 The File Page Setup item is used to setup the page for printing The File Print item is used to print the current tree The File Compare item is used to open the Compare dialog which is used to setup a comparative analysis of multiple datasets The File Extract Reads by Taxa item is used to open the Extractor dialog which is used to extract all reads assigned to a given part of the taxonom
45. m a BLAST comparison of the given set of reads to a database such as NCBI NR or NCBI NT see Section 23 for details of the file formats used MEGAN supports BLASTN BLASTX and BLASTP standard text format and BLAST XML format MEGAN can read gzipped BLAST files directly so there is no need to un gzip them although at present MEGAN processes uncompressed files much faster than compressed ones MEGAN can also parse tabular BLAST output generated using BLAST option m 8 however as this form of output does not contain the subject line for sequences matched it is unsuitable for MEGAN because MEGAN cannot determine the taxon or gene associated with the database sequence However if you add an additional column to this format containing a numerical NCBI taxon id for each line then MEGAN will parse these and use them as input Note that the reads file should be given to use the full potential of the program The BLAST file and reads file are supplied to MEGAN when setting up a new MEGAN project Both files are parsed and all information is stored in the project file The input data is then analyzed and can be interactively explored All reads and BLAST matches are contained in the project file and MEGAN provides different mechanisms for extracting them again A MEGAN project file contains all reads and all significant BLAST matches by default up to 100 matches per read in a binary and incrementally compressed format The size of such a project file
46. m identifiers of the selected nodes e The Edit Copy GO Name s item is used to copy the GO term names of the selected nodes e The Edit Find item opens the Find window which can be used to search for GO term names or IDs e The Edit Preferences submenu contains items for setting preferences The Edit Preferences Optimize View For Large Data Sets item draws the GO graph in a more optimzed way to save computation time Edges are no longer round shaped and anti aliasing is turned off The Edit Preferences Show Colored Read Assignment Table item can be used to turn on and off the heat map like coloring of the tabular listing The Edit Preferences lt Antialiased Painting item can be used to turn on and off anti aliasing The Edit Preferences Set Label Font Size item can be used to change the font size of the node labels The Edit Preferences Synchronize GO Term Selection item can be used to turn on and off the automatic focussing of nodes in the view when the user clicks on an entry in the list The Edit Preferences gt Node Coloring item can be used to change node color scheme blue red The Edit Preferences Show Node Labels item can be used to choose whether the node labels should be visible 15 4 Options Menu e The Options Select Subgraph item is used to select all child nodes of currently selected nodes e The Options Highlight Paths of Selected Nodes item is used to h
47. ntains entries from GenBank and is not non redundant It contains untranslated gene coding sequences and also mRNA sequences 8 Identification of COGs The program will attempt to map any read toa COG that is to cluster of orthologous groups of proteins see http www ncbi nlm nih gov CO0G At present this is done simply by looking for COG identifiers in the header line of the BLAST hits e g COGOO9 will be intrepreted as COG number 009 Some entries in the NR database contain such COG identifiers We assume that only references sequences of COGs are contained in the NR database but have not checked this Hence it may be necessary to run a separate BLAST comparison against the COG database after modifying the headers there appropriately so that they contain COG identifiers as described above 9 Assigning Reads to Taxa The main problem addressed by MEGAN is to compute a species profile by assigning the reads from a metagenomics sequencing experiment to appropriate taxa in the NCBI taxonomy At present this program implements the following naive approach to this problem 1 Compare a given set of DNA reads to a database of known sequences such as NCBI NR or NCBI NT 2 using a sequence comparison tool such as BLAST 1 2 Process this data to determine all hits of taxa by reads 3 For each read r let H be the set of all taxa that r hits 4 Find the lowest node v in the NCBI taxonomy that encompasses the set of hit t
48. ntion the names of variables should be three letters long A typical format string will contain some of the following variables Name type rid string rln long tid string hit long bit double exp double idy double fra long sfa long sfb long sum int interpretation Read ID Read length NCBI taxon ID Number of hits between this read and this taxon bit score of alignment expected score percent identity frame used in BLASTX hit start position of hit in source file end position of hit in source file number of reads summarized by this line A read hit definition may contain less values than there are variables in the format line In this case all trialing variables are assigned a null value To assign a null value to in variable that is not at the end of a read hit definition use the character Here is an example ofa MEGAN file Source megan data blast CreationDate Wed Mar 29 03 19 54 CEST 2006 Creator MEGAN built 10 March 2006 Format rid rln tid bit exp fra sfa sfb psc 001015_0656_2350 003500_0107_1715 005388_0322_3089 006569_0422_ 3302 008915_0625_2885 004296_0382_2957 009643_0558_2904 93 103 101 107 105 235909 32 7 4 1 2 19612521 19612874 1 113 316273 36 2 0 37 1 11739468 11739958 1 92 7460 45 4 6 0E 4 2 19781905 19782258 1 27 23 2 Full Summary and Comparison MEGAN Files MEGAN currently destinguishes between three types of text files The ContentType field may take on one of the thr
49. om and inspect the data inspector and chart tool are available The panel on the left shows exactly how many reads are assigned to a certain GO term Double clicking on a node will highlight its path in the graph A triple click will additionally highlight its child terms in the list The mouse wheel can be used to zoom into or out of the graph Clicking the right button and at the same time moving the mouse will scroll the graph view in the corresponding direction 15 1 Exploring the Read Assignments Besides the displayed graph view the GOAnalyzer window contains an information panel on the left to explore GO terms of the read assignment By default a tabular listing provides a 20 comprehensive overview of all GO terms that have been assigned with read sequences In addition to the number of the assigned reads for each data set the following columns are listed e GO Term the full name of the GO term e Specificity The specificty score of each GO term is computed as follows This value is based on the Shannon Information Content IC and on the number of annotated genes for each term as listed here http www geneontology org GO current annotations shtml filter The IC of a term reflects the frequency of gene annotations to that term or to descen dants in the sub graph of that term Terms often used for annotated gene products are assigned with a lower specificity than infrequently used terms Formulas adapted from http ww plo
50. or use the arrow keys To zoom the graph in verticial or horizontal direct press the shift key while using the arrow keys To increase the zoom factor additionally press the alt key or the control key To select a region of nodes using the mouse while pressing the shift key click and then drag the mouse in the window 12 Import Dialog The Import dialog is used to import new data from BLAST and to create a new RMA file The dialog has five tabbed panes The first tabbed pane titled the Wizard pane provides an Import wizard for creating a new RMA file The user is first asked to specify a BLAST file then a reads file and finally the name of the new RMA file to be created Once this information has been collected the user can press the Apply button to import the data The other four panes are for advanced users The second tabbed pane titled the Content pane can be used to specify whether the COG content shall be analyzed additional to an analysis of the taxonomical content The third tabbed pane titled the Files pane can be used to setup the location of files The first two items are used to specify the location of the input files to be read namely the BLAST file and the reads file The third item is used to specify the location of the new RMA file This pane provides two options The Maz number of matches per read file specifies how many matches per read to save in the RMA file A small value will reduce the size of the RMA file but m
51. ractor dialog is opened using the File Export Reads item The dialog is used to extract all reads assigned to selected taxa For any selected taxon all reads assigned to it or to any taxon below it in the hierarchy are saved to a file Use the top Browse button to add specify a file containing DNA reads in FastA format Use the button multiple times to specify multiple files Use the lower Browse button to specify the output directory Specify the file name for output in the File name field If the name contains t then the program will produce one output file per taxon and the name of the file is generated by replacing t by the taxon name Otherwise all reads are written to one file If Preserve existing files is selected the program will not overwrite existing files 21 Export Image Dialog The Export Image dialog is opened using the File Export Image item This dialog is used to save a picture of the current tree in a number of different formats see Section 23 5 The format is chosen from a menu There are two radio buttons Save whole image to save the whole image and Save visible image to save only the part of the image that is currently visible in the main viewer If the chosen format is EPS then selecting the Convert text to graphics check box will request the program to render all text as graphics rather than fonts 25 Pressing the apply button will open a standard file save dialog to determine where to save the graphics
52. read matches Otherwise if each line of the CSV file contains three strings separated by a comma the first string will be interpreted as a read id the second one as a taxon name or id and the third one will be interpreted as a bit score for this assignment MEGAN will assume that this data describes a collection of reads and their matches This data will be analysed using the LCA algorithm and the result will be displayed on the NCBI taxonomy For example assume that you have done a database search using some other method than BLAST and have obtained the following result e The read r01 matches Escherichia coli CFT073 with a bitscore of 100 e The read r01 matches Escherichia coli K12 with a bitscore of 110 and e The read r01 matches Salmonella enterica subsp enterica serovar Choleraesuis str SC B67 with a bitscore of 120 31 e The read r02 matches Caldicellulosiruptor saccharolyticus DSM 8903 with a bitscore of 90 To import this data into MEGAN so as to analyze is using the LCA algorithm produce the following CSV file r01 Escherichia coli CFTO73 100 r01 Escherichia coli K12 110 r01 Salmonella enterica subsp enterica serovar Choleraesuis str SC B67 120 r02 Caldicellulosiruptor saccharolyticus DSM 8903 90 23 7 Tree and Map Format The NCBI taxonomy is loaded by MEGAN at startup It is contained in a NCBI tree file in the standard Newick tree format The mapping from taxon IDs to taxon names is loaded by MEGAN at start
53. rences of the query The direction in which the next match is searched for can be selected using the Forward and Backward buttons The search can be applied to different targets e Nodes search all node labels e Collapsed Nodes search among the collapsed nodes and then uncollapse any found nodes e Edges search among edge labels e Reads search among the set of reads Here the whole header line of each read is searched e BLAST hits search among the set of BLAST hits Here the whole text of each match is searched e Messages search among text in the Messages window Press the From File button to load a set of queries one per line from a file If no data has been loaded into the program then it can be used to explore the NCBI taxonomy 15 GOAnalyzer Window The GOAnalyzer window enables to analyze the functional content of a metagenome using the classification structure of the Gene Ontology GO Nodes represent the GO terms whereas edges represent the relationships The read assignment to GO terms are visualized in an interactive graph view displaying all GO terms found in the data set and additionally all nodes that lie on the path towards the root node The amount of read hits per GO term in the DAG is represented with a color gradient The comparison views are the same that MEGAN uses for the taxonomical analysis pie chart heatmap meters The GO terms are organized in an interactive graph view that lets you zo
54. reshold for the bit score divided by the read length of hits Any hit in the input data set that scores less than the given threshold is ignored This is useful when the reads have widely varying lengths 24 e The Top Percentage item can be used to set a threshold for the maximum percentage by which the score of a hit may fall below the best score achieved for a given read Any hit that falls below this threshold is discarded e The Win Score item can be used to try and separate matches due to sequence identity and ones due to homology If a win score is set then for a given read if any match exceeds the win score only matches exceeding the win score winners are used to place the given read The hope is that secondary homology induced matches are discarded in the presence of stronger primary matches 19 Compare Dialog The Compare dialog is opened using the File Compare item This dialog provides a list of currently open datasets To construct a comparison select at least two different datasets and then press ok Select Use absolute counts if you want the comparison the original counts of reads for each dataset Select Normalize over all reads if you want all counts to be normalized such that each dataset has 100 000 reads Select Ignore Not Assigned and No Hits if you want all reads assigned to the two special nodes labeled Not Assigned and No Hits to be ignored 20 Extractor Dialog The Ext
55. rithms used to place reads into the taxonomy The main viewer shows disabled taxa in grey By default all environmental samples and similar taxa are disabled There are three items the Options Taxon Disabling Enable All item enables all taxa the Options Taxon Disabling Disable Selected item disables all currently se lected taxa the Options Taxon Disabling Enable Selected item enables all currently se lected taxa 13 11 6 The the Options Taxon Disabling List Disabled item lists all currently disabled items Use the Options Set Number of Reads item to set the total number of reads in the analysis By default this number is set to the number of different reads encountered in the input file The Options List Summary item produces a textual report on how many reads hit each of the nodes in the taxonomy To format is readid taxon name where the two are separated by a tab The Options List Microbial Attributes produces a textual summary of the taxon represented by the selected node The Options List COGs item produces a textual report on which reads are assigned to which COG The Options Open NCBI Web Page shows the NCBI taxonomy web page for the selected taxon The Options Inspect item is used to display the currently selected taxa in the Inspector window Double clicking on the label of a node has the same effect Tree Menu Tree menu contains the following items The Tre
56. scompbiol org article info 3Adoi 2F10 1371 2Fjournal pcbi 1000431 s3 and http nar oxfordjournals org cgi content full 35 supp1_1 D322 e Level The graph level of a term indicates the maximum path length to this term node starting at the root node If a term can be reached via multiple paths only the maximum path length is considered e Divergence only for the comparative analysis The divergence of each GO term repre sents the maximum difference in read assignments between the compared data sets Large divergences likely indicate GO terms of interest e Reads Total only for the comparative analysis The sum of reads for all data sets assigned to each GO term We now discuss all menus of the GOAnalyzer window 15 2 File Menu The File menu contains the following file related items e The File Export submenu contains items for data export The File Export Graph View is used to save the current GO graph as jpg file The File Export Table View item is used export an image of the tabular listing of the GO terms as jpg file The File Export Read Assignment item is used to export the tabular listing as tab delimited text file e The File Page Setup item is used to setup the page for printing e The File Print item is used to print the current GO graph visualization e The File Close item is used to close a window 21 15 3 Edit Menu e The Edit Copy GO ID s item is used to copy the GO ter
57. t RMA is an open format which we will describe in a separate document 23 1 The MEGAN Text File Format MEGAN also supports a line based format and each line defines either a global variable or a read hit A line starting with a is treated as a comment and is ignored Global variables should appear at the top of the file although this is not enforced Any line starting with a is expected to contain the definition of a global variable in the format name value where name can be any word starting with a letter and not containing a and value is terminated by the end of line The following global variables are generated by the parsers implemented in MEGAN 26 Source CreationDate Creator Format Algorithm Parameters ContentType TotalReads contains the location of the source comparison file This is required by the Inspector window to look up and to display the text of BLAST hits contains the date that the data was generated contains information on the program used to generate the data defines the format of all subsequent read hit lines contains the name of the algorithm used to assigned reads contains the parameters used by the algorithm is either Full Dataset the default or Summary contains the total number of reads Any line not starting with a or describes one read hit and consists of a list of values that are assigned to variables as specified by the format string By conve
58. tical Expand image vertically contract direction vertical Contract image vertically zoom selection Zoom to current selection of nodes Selection select all Select all nodes select none Deselect all nodes select leaves Select all leaves select internal Select all internal nodes select subtree Select all nodes in subtrees below selected select intermediate Select all intermediate nodes select level name Select all nodes at named taxonomical level Searching find searchtext text target Nodes Collapsed Edges ReadIDs all bool regex bool wholeword bool respectcase bool1 Find and select the next label matching the given search text replace searchtext text replacetext text target Nodes Collapsed Edges ReadIDs all bool regex bool wholeword bool respectcase bool Find and select the next label matching the given search text Reading writing and parsing synonyms and taxonomy files open synonymsfile name Open and load the named synonyms file set usesynonyms bool Use loaded taxon name synonyms when importing data load lookupdir name Load accession lookup files from the named directory set useaccessionlookup bool Use the loaded accession lookup data when importing data open mappingfile name Open the named mapping file open taxonomyfile name Open the named taxonomy file open cogsfile name Open the named COGs definition whog txt file save taxonomyfile name Save the taxonomy to the named file parse ncb
59. tuebingen de software megan see Section 3 for details Upon startup the program will automatically load its own version of the NCBI taxonomy and will then display the first three levels of the taxonomy To explore the NCBI taxonomy further leaves of this overview tree can be uncollapsed To do so first click on a node to select it Then use the Tree Uncollapse item to show all nodes on the next level of the taxonomy and use the Tree Uncollapse Subtree item to show all nodes in the complete subtree below the selected node or nodes To explore the NCBI taxonomy in a more directed fashion open the Edit Find dialog type in a part of the name of a taxon of interest and then press the Collapsed taxa target button This will request MEGAN to search for all matches to the given input and will un collapse all nodes in the tree necessary to show the matching taxa To analyze a data set of reads first BLAST the reads against a database of reference sequences such as NCBENR 2 using BLASTX 1 or BLASTP NCBENT 2 using BLASTN 1 or against one or more genome sequences using BLASTZ 5 say Then import the BLAST file into MEGAN using the File Import BLAST menu item The Import wizard will ask you to enter the name of the BLAST file a reads file containing all the read sequences in multi FastA format if available and the name of the new output RMA file Some implementations or output formats of BLAST suppress those reads for which no
60. up It is contained contained in a NCBI mapping file in a line based format in which each has three entries taxon ID taxon name and then a number indicating the size of the genome or 1 if the size is unknown 24 Command Line Options and Mode MEGAN has the following command line options t lt String gt default tree file i lt String gt default ID to name mapping file fc lt String gt default COGS definition file f lt String gt default MEGAN file fs lt String gt default Synonyms file ld lt String gt default Accession lookup directory p lt String gt default Megan def Properties file m lt int gt default 0 minimum score g lt switch gt default true gui mode w lt switch gt default true show message window x lt String gt default Execute this command at startup non gui mode only V lt switch gt default false show version string S lt switch gt default false silent mode d lt switch gt default false debug mode s lt switch gt default true show startup splash screen h lt switch gt default false Show usage Launching the program with option g will make the program run in non GUI command line mode first excuting any command given with the x option and then reading additional commands from standard input Please be aware that the command line version of the program uses the same properties file as the interactive version
61. y The File Extract Reads by COG item is used to extract all reads assigned to a given COG The File Import CSV item is used to import data from a comma separated CSV file 10 11 2 The 11 3 The The File Tools submenu contains menu items for loading alternative tree and mapping files The File Properties item displays a summary of the current data This window also shows which versions of the NCBI taxonomy NCBI microbial attributes and COGs are used by the program The File Close item is used to close a window The File Quit item quits the program Under MacOS this item is contained in the MEGAN menu Edit Menu Edit menu contains the usual edit related items The Edit gt Cut item is used to cut text e g when editing the label of a node The Edit Copy item is used to copy text or to copy the current tree as an image The Edit Paste item is used to paste text The Edit gt Edit Node Label item is used edit the labels of nodes The Edit Edit Edge Label item is used to edit the labels of edges The Edit Format menu item opens the Format window that can be used to change the font size line width and color of nodes and edges The Edit Find item opens the Find window which can be used to search for taxa and reads The Edit Find Again finds the next occurrence of a search string The Edit Preferences submenu contains items for setting preferences The Edit Preferences
62. zing attributes of the taxa The Window Command syntax item lists all valid commands The Window Enter a command item can be used to execute a command The bottom of the Window menu contains a list of all open windows 15 118 MEGAN Menu Under MacOS there is an additional standard menu associated with the program called the MEGAN menu As usual this contains the Window About and File Quit menu items 11 9 Tool Bar The Main window provides a tool bar containing buttons that provide short cuts to some of the menu items associated with the window These are the File Open File gt Print Layout Expand Contract Expand Vertical Layout Expand Contract Contract Vertical Layout Expand Contract Expand Horizontal Layout Expand Contract Contract Horizontal Layout Fully Contract Layout Fully Expand and Edit Find items 11 10 Popup Menus The Main window provides three different popup menus that are activated by right clicking on a node an edge or the background in the Main window If are using a single button mouse under MacOS then please control click to access these menus The popup menu that is opened when a node is right clicked on has the following items e The Inspect adds the selected node to the Inspector window and opens that window if necessary e The Edit Node Label opens a dialog to change the label of the selected node e The Copy Node Label copies the node label to the system

User Manual for MEGAN V3.8 - HIGH THROUGHPUT SEQUENCING

Contents

Download Pdf Manuals

Related Search

Related Contents