BYU Phamerator Instructions
Contents
1. 1 etc is also critical locus tag JIMMER1 1 is one of the most important features in the GenBank file If you cannot see a locus tag in the file then you cannot import it into Phamerator You will need to use DNA Master or another program to add phage specific locus tags and then export a new GenBank file translation The first letter inside quotations after the translation section should be an M for each gene If it is not you may encounter an error in Terminal as you try to import the phage into Phamerator When you export files from DNA Master you must select Bacterial and Plant Plastid Code This results in transl_ table 11 being displayed next to each gene This helps the phage genome import correctly into Phamerator 3 Using DNA Master to prepare GenBank files from NCBI for Phamerator If GenBank files from NCBI are missing any of the above features they need to be processed using DNA Master before they can be successfully imported into Phamerator DNA Master only runs on Windows a Download DNA Master It can be found at http cobamide2 bio pitt edu computer htm Make sure you have the most up to date version by running DNA Master as an Adminstrator right click on the icon choose Run as Administrator After it starts choose Help and then Update DNA Master b Paste the accession number for the phage you want to download into the bottom left corner of DNA Master Click the red checkmar2. 3 10 in Cdd pn out Cdd threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title PRK v 6 00 in Prk pn out Prk threshold 9 82 scale 100 0 dbtype rps index true Add conserved domains to gene products in Phamerator database run in Applications phamerator dev phamerator plugins directory eddbearch py s a cdd Ob Cdd ssas COdLastlay query Lasta 152
3. Jimmerl Unclassified REFERENCE 1 bases 1 to 54312 AUTHORS Merrill B D TITLE Direct Submission JOURNAL Submitted 25 JAN 2013 FEATURES Location Qualifiers source 1 54312 organism PL Jimmer1 mol type genomic DNA note complete genome 26 463 gene 1 locus tag JIMMER1 1 26 463 gene 1 locus tag JIMMERI1 1 note Terminase small subunit codon_start 1 transl table 11 product gp1 translation MKLTPKQQOAFADYYIQIGNATEAARKAGYSDKTAKEVGYENLTK PHIKAY IDE RMAVKDAERIASQDEVLEFLTNVMRGKVTEKI PLGLGMGEOQGLVKNELO GKDRIKAABFLIGKRYGLWVEKVNLDGDLAVTIIDDIGVDDEEG Figure 9 Example of a GenBank file containing features that are necessary for Phamerator PL Jimmer1 is the organism name This is the name that will appear in Phamerator as the name of the phage However if there are any spaces between the quotation marks in this area Phamerator will only import the last word after the last space and use that as the title of the phage IMPORTANT As you prepare each GenBank file and modify the organism names as necessary keep track of the phage names in an Excel spreadsheet with the names in the far left column Gene and CDS are necessary for each gene or protein described by the GenBank file Occasionally you will find GenBank files that do not contain the gene feature If the gene feature is missing then Phamerator will likely not import the file correctly You should process the file with DNA Master first The gene
4. account for this to work To check or change this click in the top right corner choose System Preferences and then User Accounts Using this same procedure install the following programs one at a time by typing the code below and press Enter For best results install programs sequentially down the left column and then down the right column apt get install git sudo apt get install python pygoocanvas apt get install python sudo apt get install python webkit apt get install perl sudo apt get install python mysgldb apt get install pyro sudo apt get install mysql server apt get install clustalw sudo apt get install python pip apt get install clustalo sudo pip install numpy apt get install curl sudo pip install biopython apt get install perl doc sudo apt get install python biopython apt get install python dev sudo apt get install python pp apt get install ncbi blastt sudo apt get install blast2 apt get install bazaar When installing mysql server you will be prompted to enter a root password Don t forget this password While installing you may have to type y for yes and then press enter Outside Terminal click on the Folder or Filing Cabinet icon see Figure 8 This is your Home folder Inside the Home folder right click and choose Create new folder Type Applications and press Enter In Terminal navigate to the Applications cd Applications folder and
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6. the directory you specified importseq will be imported Type Y and press Enter to continue f There are a few errors you may encounter If you encounter an error Terminal will ask you whether or not you want to proceed Type n for no and press enter and the database should remain unchanged If a phage genome is imported and the Phamerator user interface indicates that the genome has zero genes delete the phage and try again step V 6 j Common errors include Invalid start codon usually not an issue just press Y and continue importing Translation from GenBank file does not match translation computed by Phamerator usually not an issue just press Y and continue importing This is caused when the translation table is something other than Bacterial and Plant Plastid or transl_table 11 GeneID already in use the locus tag for this gene is not unique among all genes in the database Exit and generate new unique locus tags for this genome using DNA Master GeneID truncated your organism name and locus tag are too long Shorten one or both using Find and Replace in the Ubuntu text editor g After the import process is completed you will receive a prompt asking you whether you want to delete the ClustalW and BLAST scores Type N for no and press Enter 144 To see if the GenBank file imported correctly type Phamerator and press Enter Type in your username root password your my
7. what you want to name your database If you want to create a MYSQL database on a networked MYSQL server not on your computer use this command mysql u root p h server univ edu e create database testphage Substitute server univ edu with the address of the networked MYSQL server The h argument and the server name can be added to any of the commands described below for databases hosted on a server Next we need to customize the blank database for use with Phamerator by first navigating in Terminal to the sql folder of the Phamerator directory cd Applications phamerator dev phamerator sql1 from the home folder Enter the following command mysql u root p testphage lt db schema sql Substitute testphage with the name of the database you created GenBank files can now be imported into this database Proceed to step 2 To delete databases e Log in to MYSQL using the command mysql u root p press Enter type the password and press Enter Enter the command drop database testphage 140 Substitute smegphage with the database you want to delete To export or back up a database into a single file f Use the following command type on one line mysqldump u root p testphage default character set utf8 result file testphage sql Substitute testphage with the name of the database you want to export into a file and testphage sql with what you want to call t
8. Builder4 to fix existing phams rather than delete the phams and build them from scratch After you are finished modifying your database re run ClustalW BLAST phamBuilder4 and cddSearch Your modified database is ready to use VI HOW TO USE THE PHAMERATOR USER INTERFACE TO EXPLORE THE DATABASE 1 Displaying linear genome maps a eS Start Phamerator by typing Phamerator within the Applications phamerator dev phamerator directory and enter your username password server address and database name Phamerator will start Click on the Phages tab While holding the Ctrl button down click on all of the phages you want to appear in the map Click the Map button at the top of the screen You will see lots of code fly by in Terminal and eventually a new window will open displaying the genome map You can zoom in and out or reorder the map by clicking on the phage name and dragging it up or down You can align the maps by clicking on one gene in each genome and choosing align left or align right You can save this map by clicking File then Save As and choose a PDF file Figure 12 below shows what a linear genome map of five phages looks like 148 s m 5 m sm H za see H am Fm E gt F as E wo a game tei me me weg me sna sn r ee gun e sm gg EE m CD C MEE se D 2 ai on len n ey MA Bun a oo GED O So e iR
9. II HI Sa deme ae ce ae IV NO m aie a ak ae ee S VI eee VII VIII IX Setting Up Phamerator and Creating Usable Databases for Bacteriophage Comparative Genomics Bryan D Merrill Andy T Ward Julianne H Grose Sandra H Burnett Table of Contents INTRODUCTION HARDWARE REQUIREMENTS HOW TO SET UP A UBUNTU VIRTUAL MACHINE Download Ubuntu 14 04 LTS Install the latest version of VirtualBox or other virtualization software and create a virtual hard drive Install Ubuntu on your new virtual hard drive Install the VirtualBox extensions package Guest Additions Install Ubuntu Updates Learn to use basic Terminal commands HOW TO INSTALL PHAMERATOR AND OTHER NECESSARY PROGRAMS Use sudo apt get to install software from the Internet Start Phamerator and download BLAST HOW TO BUILD A PHAMERATOR DATABASE Prepare a new SQL database Collect GenBank files and determine 1f they are ready to import into Phamerator Using DNA Master to prepare GenBank files from NCBI for Phamerator Using DNA Master to import FASTA files auto annotate them and generate GenBank files for Phamerator Using DNA Master to create a GenBank file Import GenBank fixed files into Phamerator Adding cluster designations to the Phamerator database Starting Phamerator Perform ClustalW computations on the Phamerator database Perform BLAST computations on the Phamerator database 11 Wa 13 14 15 Check the progre
10. Jimmer1 gbf Right click on each file and change the file to Jimmer1 gb fixed Transfer one of the gb fixed files to the importseq folder c Open Terminal Navigate to the phamerator directory This can be done by entering the command cd Applications phamerator dev phamerator d Run the import sequence script by entering the following command and pressing Enter Phamerator will ask you whether you want to import all the contents of the folder so type y for yes and press Enter Replace testphage with the name of the database you want to receive the GenBank files phamerator manage db py u root p s localhost d testphage i 5 jf TMporreea You will be prompted for your MYSQL server password see step V 1 b This is what each part of this command means phamerator manage db py is the program used to import the sequences u tells Terminal the next thing you type is the username root is the username p tells Terminal to ask for your password s tells Terminal the next thing you type is the server address Use Localhost ifrunning a local database d tells Terminal the next thing you type is the database name test phage is the example database name i tells Terminal the next thing you type is the location of the sequence to import importseg tells Terminal to go down one directory import all of the GenBank files in the folder named importseq e Phamerator will warn you that all phages in
11. MERATOR DATABASE To build a Phamerator database containing phage genomes you will first need to collect the files from GenBank and put them in a folder within your virtual machine GenBank files can be generated by DNA Master http cobamide2 bio pitt edu computer htm or can they can be downloaded from NCBI http ncbi nlm nih gov Since Phamerator uses GenBank files to build the phage database the GenBank files need to include certain features If the GenBank files are missing features they will first need to be processed using DNA Master Preparing GenBank files for Phamerator using DNA Master is a complicated process that requires some troubleshooting so follow these instructions closely Specific tips for issues that may arise are covered in section 6f Obtain Phage Format Format Database Files Correct Import to database Fix using DNA Master or other program 1 Prepare a new SQL database a Create a new blank MYSQL database using the following command in terminal replacing testphage with the name you want for your database mysql u root p e create database testphage Press Enter You will be prompted for a password which is the password you created when you installed MYSQL Server see step IV 1 b This is the same password you will enter each time you are prompted throughout the rest of this documentation u stands for username p prompts for the password Substitute testphage with
12. NA Master to create a GenBank file a Before beginning this process you can choose to hide text in the notes box for each gene To hide all of these from the resulting GenBank file click on the small drop down arrow and choose Privatize all Everything inside the lt private gt and lt private gt designation will not appear in the notes section of the GenBank file To create the GenBank file click on the Tools menu in DNA Master and choose Submit to GenBank Click Add and navigate to the DNAMS file you have saved Under the Description tab the Locus field needs to be no longer than eight characters It is usually the accession number Paste this into the Accession field as well The Genetic Code field should be Bacteria and Plant Plastid code Under the Organism tab fill in the Organism field with the phage name remember Phamerator only imports the last word of whatever is in this field if it contains any spaces and the Lab Host with the host of the phage Under the References tab click Add Under the Process tab check Export Flat File Include gene features Include locus tags and Restrict to a single reference Click Process single project and save the resulting GenBank file as phage _name gb fixed or choose Process all listed projects and choose a folder where GenBank files for all phages ready for processing wi
13. Start your virtual machine by clicking the Start icon Shared Clipboard gt with the green arrow and log in Drag n Drop gt c After you are logged in click Devices at the top menu and choose Install Guest Additions See Figure 7 ay Sa d Click Run on the box that pops up Enter your Seer ree password in the next box and click Authenticate to gf Install Guest Additions Host D install guest additions The box with white text and a purple background that pops up is called Terminal This is the window where most of the functions of setting up Phamerator will be performed e When the guest additions are finished installing the bottom line of text in the Terminal window will say Press Return to close this window Press the enter key Figure 7 Install Guest Additions 5 Install Ubuntu Updates a Click on the Dash icon indicated by the green arrow in Figure 7 b Type Software Updater and click on it to run it c Install all available updates and restart Ubuntu when prompted 6 Learn to use basic Terminal commands a Navigate to the Dash icon marked by the green arrow in Figure 7 and type in Terminal Click and drag the Terminal icon and drop it underneath the Folder icon shown in Figure 8 beneath the Dash Home icon Click on the new Terminal icon to launch Terminal c To communicate with the computer through Terminal you need to use a unique lan
14. cal user interface You are now ready to use your knowledge of phage genomics and molecular biology to begin an in depth analysis of these phages VIII TROUBLESHOOTING AND FREQUENTLY ASKED QUESTIONS For errors and problems that you are unable to fix contact Bryan Merrill brymerr921 gmail com Andy Ward andytward9 gmail com Scott Carlson scocarl ls byu edu or Dr Sandra Burnett sandra h burnett gmail com When starting Phamerator a popup box appeared instead of the main Phamerator window Password was typed incorrectly Click the red X to close the popup window and start Phamerator again Phamerator unable to import a file because the locus tag is non unique Use DNA Master to create new locus tags see section V 2 How to check if a database was created properly Open the MySQL Workbench and click the black arrow next to your database name then click Tables right click on the phage tab and click Select Rows If everything imported correctly all phages should be listed 150 IX Install Phamerator dependencies can be done in any directory APPENDIX OF PHAMERATOR TERMINAL COMMANDS Sudo apt get Install Grt sudo apt get install python sudo apt get install perl sudo apt get install pyro sudo apt get install clustalw sudo apt get install clustalo sud apt Get anstall curl sudo apt get install perl doc sudo apt get install python dev sude ap
15. cm a SS 6 Z z Me 7 7 10SS 2 a 2e106 0 0 D O 6e 252e 19835122 H H i z are x elie fi H iA x f i see eel i i H soe H os z sm a0 HPE see mas 5 a mw gS n 5 H ns fear foe ng mem neg mm gi Peis mmeig sa gt ae m a ew i vn o GED o AOE ip sfn Gen i ig ED oo E we af GG O Ea HORTA ED mi m A ca z k 7 E ATUPRITTORATTTRSTTTONTTT SET TINETA ETTEN TERS TION n i rra N m i rmm r Dual rm r F i mT mm UT TVRATITIGETTLSETT ETEEN ETETEA TTA TTA ETTET N imum r i ii L imum r i D ii a CD 10L 2e 31 6e 47 92 06 22 06 a 2 n a i a gan e p mn iF a a we i me gg pipih h i ma ma iii fini mn Pa Eoi af Caf eof r Jaa wie a we ES ee CEG Eee me s a A C25 3g waban CDi IDO FGF OC wa Faa C mw IOHCE SPOR OR H mma lem J E e a 8 om 3 E Ka E Gn E w L Ic T fi ial fF fe Tt C EX _ k E EEE RN UT TR RSNA GS 3 a a 8 EC 2 ae Ge O z8 Figure 12 Linear Genome Map for Five Phages Displaying phamily circles a Click on the Phams tab b Click on one pham in the list c Click on the Phamily Circle button When the window appears you can zoom in and out and save the file as a PDF d Blue connecting lines represent linkage by more than 32 5 ClustalW similarity and red connecting lines represent linkage by BLAST e values less than 1e 50 Exporting Pham and C
16. e circular Version Ubuntu 64 Dit mune z icon that says New Type a name for your new virtual machine such as Ubuntu Phamerator Choose Linux and Ubuntu 64 bit Click Next See Figure 2 d Choose how much RAM your virtual machine will have access to Give it up to half of what is installed in the computer Click Next e Choose Create new virtual hard drive now and click Create In the window that pops up choose VDMK Virtual Machine Disk and click Next f Choose Fixed Size and click Next g Don t change the name of the virtual hard drive file but change the size of the virtual hard drive About 20 00 GB will be sufficient if you are only viewing databases or are not adding conserved domains Otherwise choose 80 00 GB Click Create h To allocate more processor cores to your new virtual machine choose Settings then System then the Processors tab i To enable 3D acceleration check that option under the Display tab Name Phamerator Figure 2 Name and operating system of new Virtual Machine 136 Install Ubuntu on your new virtual hard drive rae a Start VirtualBox if it is not running click once on your new virtual a op gt machine and click the Settings button at the top A new window will New Settings Start open with options on the left like you see in Figure 3 b Click Storage Click on Empty next to t
17. e inside Ubuntu or else the file needs to be moved there later It is a very large file 3 GB c Place this newly downloaded file inside the phamerator dev directory d Make anew folder called cdd_ db 147 L g h Open terminal Navigate to Applications phamerator dev and run the following command Yov ll see a ton of lines of code fly by and will know when it s done when a fresh command line appears In terminal navigate to Applications phamerator dev cdd_db Type the first command listed below to set up the CDD Press enter When it is done type the second command press enter and wait until it is done Do this for all six makeprofiledb title SMART v6 0 in Smart pn out Smart threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title Pfam v 26 0 in Pfam pn out Pfam threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title COG v 1 0 in Cog pn out Cog threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title KOG v 1 0 in Kog pn out Kog threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title CDD v 3 10 in Cdd pn out Cdd threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title PRK v 6 00 in Prk pn out Prk threshold 9 82 scale 100 0 dbtype rps index true 1 You re done creating the local CDD Now you can run the cddSearch py script and identify conserved domains in all of the genes in your Phamerator database 14 Using the Co
18. enerate jobs that it will feed to the other window the client The client Terminal window will perform computations and feed the back to the server Terminal window which inserts any significant results into the Phamerator database Each job compares one gene product to every other gene product in the database a AR C In terminal navigate to the Applications phamerator dev phamerator directory Type the following command and press Enter The number following i indicates how many instances of the client you want to run Choose 1 for most computers or 2 for a very fast computer Make sure the 1 True is actually a lowercase L Substitute testphage for your database phamServer InnoDB py u root p s localhost n localhost ad testphage 1i 1 1 True a clustalw Open a second Terminal window by right clicking on the Terminal icon and choose New Terminal Window Navigate to the Applications phamerator dev phamerator directory type the following command and press Enter phamClientOmega py u root p n localhost If you specified that you want to run two instances open a third Terminal window navigate to the Applications phamerator dev phamerator directory and enter the above command WARNING This process can take a VERY long time If you need to pause the process and resume a different time press Ctrl C on your keyboard in each client Terminal window first followed by the server window a
19. guage Terminal operates the same way you would operate your computer Instead of clicking on things you type commands into Terminal and press Enter Table 1 compares how you perform basic tasks using a mouse with how you perform those same tasks by typing commands in Terminal Mac OS press Enter cd means Change Directory cd Go up a folder level Click the Back button Display the contents of a folder Double click on the folder ls ls means list Start a program called Phamerator Double click on the Within the folder containing Phamerator Phamerator icon type Phamerator means run Table 1 Tasks to perform for setting up Phamerator in Terminal 138 IV HOW TO INSTALL PHAMERATOR AND OTHER NECESSARY PROGRAMS 1 Use sudo apt get to install software from the Internet a Termine File Edit View F OT B g a E l NMnnDNnNnNnNnNnNNANA NN VN MN ar O O O O O O O O O O To install programs you will enter a command in Terminal see Figure 8 pm a So a fa sear Cn bryan BMerrill ubuntu yan BMerrill ubuntu sudo apt get install giti Figure 8 Terminal window with command to install git The sudo argument gives administrator privileges to the command that follows The password you are prompted for is the password for the Ubuntu user account you are currently logged into see step III 3 h and Figure 6 Your account needs to be an Administrator
20. he CD logo under Controller IDE then click the CD logo on the right shown by red arrow in Figure 4 Choose Choose virtual CD DVD disk file and navigate to the Ubuntu iso file called ubuntu desktop 1so Click Open General Click Ok E System Storage Tree Attributes f CD DVD Drive Display E Live CD DVD C Storage Figure 4 Adding the iso file to the Ubuntu virtual machine Audio c Click on your virtual machine and click the Start button with the green Network arrow Ea d A new window will pop up and after a few seconds you will see a list of Serial Ports languages on the left an option that says Try Ubuntu and an option F USB that says Install Ubuntu Click on Install Ubuntu e Inthe next window click the checkboxes that say Download updates wi Shared Folders while installing and Install this third party software Click Continue f The next step will look like Figure 5 Choose Erase disk and install Ubuntu and click Continue A WARNING This will NOT erase the hard drive on your computer to install Ubuntu You are Figure 3 Options found in Settings menu only installing Ubuntu inside the virtual hard drive you created which 1s really just a file You will NOT lose any of your data by choosing this option Install Installation type This computer currently has no detected operating systems What wou
21. he file Just make sure it has a sql at the end You can compress the SQL file so it can be emailed Right click on the SQL file and choose Compress To Import Phamerator databases from an SQL file h 1 J k Create a new database step V 1 a Import the db_schema sql file step V 1 d In terminal navigate to the folder where your dumped sq file is Enter the following command MySql u TOGE p detault Character set urio testphage lt filename sql Substitute testphage with the name of the database you created and filename sql with the name of the SQL file you are trying to import 2 Collect GenBank files and determine if they are ready to import into Phamerator a Inside the virtual machine download GenBank files from NCBI and rename each file with the correct phage name If you did not save the files from NCBI in your Ubuntu virtual machine you can email the files to yourself use Dropbox or click on the VirtualBox Devices menu option go to Drag n Drop and choose Bidirectional which should allow you to drag files from one desktop to the other Examine each file to see if it is ready to be imported into Phamerator or if it will need modification Figure 9 highlights features of the GenBank file that are important to Phamerator 141 LOCUS Jimmerl 54312 bp linear ENV 25 JAN 2013 DEFINITION Jimmerl ACCESSION VERSION KEYWORDS ENV SOURCE Jimmerl ORGANISM
22. k and then choose Save c The gene feature of the GenBank file is populated by the information found in the name field in DNA Master and the locus_tag feature is populated from the Locus Tag field in DNA Master You can automatically reassign gene numbers and locus tags by clicking Validate To reassign both 142 under the Control tab leave all boxes except Label default products checked Type your new locus tag prefix in the appropriate field Under the Numbering tab make sure both fields say 1 If you don t want to reassign locus tags leave only Assign Names checked WARNING Changing gene numbers so they are different than the original ones will make it difficult to compare gene numbers in Phamerator with gene numbers in the published genome Occasionally phage genomes are not numbered sequentially You may have to edit all of the name and locus tag fields manually if you wish to preserve the original gene numbers Phages are often arranged in a manner that is inconsistent with other similar phagess If you wish to rearrange the phage genome so it begins at a new location go to the top of DNA Master and choose Genome Rearrange and click on the Rearrange tab Include all annotation except Original Coordinates and type in the base pair where you want the new genome to begin Make sure this doesn t interrupt any genes and click Rearrange In the new wi
23. ld you like to do Erase disk and install Ubuntu Warning This will delete any files on the disk Figure 5 Erase disk and install Ubuntu g As Ubuntu is installing you can enter information about your computer i Enter your location by typing in your city state and country ii Choose the English US Install keyboard layout h Enter the information displayed in Figure 6 including your name a name Your name for your computer use no spaces a PEE E a username lowercase no spaces a The name it uses when it talks to other computers password and choose Require my ee password to log in Click Continue WARNING Write down your username and password somewhere in eee case you forget it 7 Encrypt my home folder i Be patient as Ubuntu finishes installing When it is done click Restart Now j When prompted press Enter to finish restarting the virtual machine Who are you Choose a password Password Confirm your password Confirm password Log in automatically Figure 6 Enter information about your virtual machine 137 4 Install the VirtualBox extensions package Guest Additions Ubuntu Phamerator Running Oracle VM VirtualBc a With your virtual machine shut down navigate to the Help universal extension pack and double click on it Follow CD DVD Devices gt the directions to install it USB Devices gt b
24. ll be created All GenBank files to be imported into Phamerator need to end in fixed 143 Import GenBank fixed files into Phamerator A good method for building a Phamerator database is importing one GenBank file at a time into a test Phamerator database If it works move the GenBank file to a folder for correctly formatted files If it doesn t work move it to a folder for incorrectly formatted files You ll then need to troubleshoot it and make a new file using DNA Master Before you try to import it again you ll need to delete the phage from the test database step V 6 or delete and recreate the database step V 1 e WARNING Once you import a sequence into Phamerator sometimes you cannot delete it without A deleting everything in the database Therefore if you encounter any of the errors described below be sure you really want to import the sequence a Click on the Folder icon and navigate to the phamerator dev folder Inside the phamerator dev folder create a folder called importseq one called importseqworked and one called importseqfail You will transfer GenBank files one at a time to the importseq folder to import into Phamerator If it works it goes to the importseqworked folder if it doesn t move it to the importseqfail folder b Navigate to the folder where your GenBank files are located The file name will be something like Jimmer1 gb or
25. luster Tables a Click on the Phages tab b Go to File gt Export Pham Table This is a csv file which can be imported into Excel using Data gt Import from Text and then finding that file With a little modification it is a very powerful tool If the conserved domains have not been added using cddSearch then all cells in the domains column will say None If cddSearch has been run then any cells in the domains column that say None have no identifiable conserved domains Exporting genomes genes or proteomes a Select phages in the Phages tab Click File and choose whether to export a single FASTA file containing all of the genomes genes or proteins belonging to the phages selected b Select a pham in the Phams tab Click File and choose whether to export a single FASTA file containing the gene or protein sequences for the members of the pham Accessing the Phamerator Quick Start User Manual a Fora description about other things you can do with Phamerator go to Help and click on User Manual 149 VIL CONCLUSION Congratulations Your Phamerator database is now functional You have now learned how to Create a Ubuntu virtual machine Install Phamerator and the software it needs to run Create a new Phamerator phage database by importing fixed GenBank files processing them using ClustalW and BLAST assign phamilies and identify conserved domains Operate the Phamerator graphi
26. merator directory phamClientOmega py u root p n localhost Start server for processing BLAST jobs run in Applications phamerator dev phamerator directory phamserver TnnoDBypy u toot p s Localhost n localhost d testphige 1 4 l True a blast Start client for processing BLAST jobs run in Applications phamerator dev phamerator directory Plestclient py u root P r localhost a 4 3742 BLAST Gin d blastdata Start progress window for ClustalW or BLAST jobs run in Applications phamerator dev phamerator directory s phamSetver progress py U Toot p s localhost d vestphage r 10 a clustalw Build phamilies phamBuilder4 py u root p s localhost d testphage c 0 325 b le 50 After downloading the cdd tar gz file conserved domain database extract the contents run in Applications phamerator dev directory tar savat Cddstar soz cad db Build CDD database run in Applications phamerator dev cdd_db directory makeprofiledb title SMART v6 0 in Smart pn out Smart threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title Pfam v 26 0 in Pfam pn out Pfam threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title COG v 1 0 in Cog pn out Cog threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title KOG v 1 0 in Kog pn out Kog threshold 9 82 scale 100 0 dbtype rps index true makeprofiledb title CDD v
27. n data to Phamerator databases 100 GB of free hard drive space is recommended III HOW TO SET UP A UBUNTU VIRTUAL MACHINE Skip this section if the computer is running Ubuntu natively 1 Download Ubuntu 14 04 LTS a Visit the link http www ubuntu com download desktop b Download 64 bit Ubuntu the top option 2 Install the latest version of VirtualBox or other virtualization software and create a virtual hard drive VirtualBox platform packages The binaries are released under the terms of the GPL version 2 o VirtualBox 4 3 12 for Windows hosts gt x86 amd64 VirtualBox 4 3 12 for OS X hosts x amp 6 amd64 VirtualBox 4 3 12 for Linux hosts VirtualBox 4 3 12 for Solaris hosts x86 amd64 VirtualBox 4 3 12 Oracle VM VirtualBox Extension Pack All supported platforms Figure 1 Screenshot from VirtualBox download page a Download the latest version of Oracle VirtualBox for your operating system as well as the universal extension pack from the Name and operating system Please choose a descriptive name for the new virtual machine and following website select the type of sa coi intend we install ses TH name you choose wi used throughout VirtualBox to identity this https www virtualbox org wiki Downloads ara see Figure 1 b Install VirtualBox and the extension pack you downloaded in step 2a then start VirtualBox kia E oooasaassasaasssIsIsuSl c After VirtualBox opens Click the blu
28. n phamBuilder4 later and it will update and not renumber the phams from scratch After you have one folder containing all of the fixed GenBank files that have been successfully imported into Phamerator using your test database create a new database and import these files step V 6 d 7 Adding cluster designations to the Phamerator database a Cluster designations can be used to group phages based on the host they infect phage morphology or other user defined characteristics In the spreadsheet you made of each phage name Section V 2 b assign a cluster in the cell immediately to the right of each phage Cluster names can be up to 5 characters long and contain only letters and numbers Phage that are singletons that don t have a cluster assignment should have NON as their cluster You will need at least one phage in the NON cluster or your pham circles will not display The table will look like this Emey B To generate the cluster file you will need to import into Phamerator select the two column table from top to bottom and copy it Paste it into Notepad or a basic text editor Click File Save As and type a file name Save it as a csv file Place it inside a folder named cluster that you create inside the phamerator dev directory In Terminal navigate to phamerator dev phamerator plugins and perform the following command update cluster py u root p s localhost d test
29. nd the process will stop When you start it again it will pick up where it left off When this long process is completed you will notice that the first window repeatedly displays No work units available Sleeping You can safely end the processes in each window using Ctrl C and move on to the next step 10 Perform BLAST computations on the Phamerator database a The second process that needs to be run is BLAST This process also requires multiple terminal windows which can be opened by right clicking on the Terminal icon and choosing New Terminal This process also requires some setup Note that this process does NOT link to the internet or use the 146 BLAST feature found on NCBI It runs locally comparing only the data found in the database You can run as many instances of BLAST as you have processor cores assigned to the virtual machine For each instance you will need to create separate folders in the phamerator dev folder for BLAST data Name them blastdata blastdata2 blastdata3 etc and make as many as you have cores on the computer From within the phamerator directory type the following command in one Terminal window and press Enter To change the number of instances substitute that number for 4 in the following command phamServer InnoDB py u root p s localhost n localhost d testphage 1i 4 1 True a blast b Inthe second window navigate to Applications phamerator dev phamerator ty
30. ndow uncheck Assign new locus tags as well as everything else on the right side of the window Leave the checked features checked Click Parse Save the DNA Master file You can also reverse complement the phage genome in a similar manner by choosing DNA at the top of the DNA Master window click Convert and choose Complement and edit the Parse box as above If any of these steps do not produce the desired results before rearranging or complementing click on the documentation tab and click recreate documentation You can also do this after rearranging or complementing When your DNA Master file is ready choose File and Save as DNAMS file and save it to a specific location 4 Using DNA Master to import FASTA files auto annotate them and generate GenBank files for Phamerator a b In the top menu choose File Open and choose FastA Multiple Sequence File Navigate to your FASTA file and click Open If you have multiple phage genomes in your FASTA file click on the one you want to use In the bottom right corner choose Export and click Create sequence from this entry only In the DNA Master menu choose Genome and then Auto Annotate Under the Tags amp Comments tab change the Prefix to the phage name or whatever you want the locus tag prefix to be Click Annotate and save the resulting DNA Master file 5 Using D
31. nserved Domain Database CDD Search a To identify conserved domains in each gene product in the Phamerator database first make a folder in the phamerator dev directory called cddfasta Next run the following command in Terminal from the Applications phamerator dev phamerator plugins directory You will then be prompted for the username password server and database Cddsearch py es a Acada dbyCdd s s cddtasta query fasta This command can be run at the same time as BLAST or ClustalW ClustalW and BLAST cannot be run at the same time Please be aware that aside from what happens immediately after you start this script you will NOT see anything happen in the Terminal window until the process is completely done Just let it process until you see a new blank line appear This could take a few hours 15 Modifying the Phamerator Database a You can add additional phages or remove phages in the Phamerator database without reassigning all of the phams or deleting the BLAST and ClustalW scores When adding or removing phages you will be prompted to delete all BLAST or ClustalW scores or to leave them alone If you don t want to recompute all of these scores make sure you don t delete them specify N for no Also if you want to keep the phams make sure you do not delete pham assignments when prompted even though Phamerator says that removing phages invalidates pham assignments You can always re run pham
32. pe the following command and press Enter Jd blastclient py root p n localhost a BbAST bin d blastdata c Open a third fourth and fifth window navigate to the correct directory and use the exact same command as above except for the final argument bolastdata In the third window change blastdata to blastdata2 inthe fourth window to blastdata3 etc d This process will also tell you when it is done When it says Sleeping press Ctrl C to end it 11 Check the progress of ClustalW or BLAST Since ClustalW and BLAST can take a long time complete you can open an additional Terminal window and run a script to display the progress To show the ClustalW or BLAST progress meter do the following a Navigate to Applications phamerator dev phamerator b Run the phamServer progress py script by using the following command phamServer progress py u root p s localhost d testphage r 10 a clustalw c Replace testphage with the name of your database and a with blast if you are running BLAST d The number after the r argument indicates how often in seconds you want to refresh the progress bar The current command will refresh it every 10 seconds Click Cancel to close the progress bar window This will NOT affect ClustalW or BLAST processing You can close and reopen this window as you wish by running the script above e When ClustalW or BLAST is finished processing the p
33. phage update cluster cluster phage cluster csv Substitute testsphage with the name of the database you are using and phage cluster csv with the name of your cluster file 145 Imported Performing Database ClustalW Creating Adding Conserved Performing BLAST Phamilies Domains Completed Database 8 Starting Phamerator After adding phages or clusters to the Phamerator database but before performing computations you can start Phamerator to make sure the phages imported correctly You will only be able to access the data in the phages tab until all computation steps are completed a To start Phamerator navigate in Terminal to Applications phamerator dev phamerator Type Phamerator You will be prompted for username password server and database name If you are running a local database you can use root for username the MYSQL server password localhost for server and type the name of the database you want to load for database If a popup window appears prompting you to re enter the password you typed incorrectly in the Terminal window Click on the red X and start Phamerator again 9 Perform ClustalW computations on the Phamerator database These computations require a lot of computing power and may take a very long time Be prepared for this ClustalW and BLAST are run using a server client interaction One Terminal window the server will look through the database and g
34. repare MYSQL database for Phamerator use From directory where SQL file to import is stored run mysql u root p testphage lt filename sgl 151 Import GenBank files into Phamerator database run in Applications phamerator dev phamerator directory j phamerator manage db py U toot p s localhost d testphage i e7 IMPOR Sg Remove genomes from Phamerator database run in Applications phamerator dev phamerator directory phamerator manage db py u root p s localhost d testphage remove name TAL7A E Add cluster designations to phages in Phamerator database run in Applications phamerator dev phamerator plugins directory Yupdate cluster py u TOOL p s localhost d testphage update cluster cluster phage cluster csv To start Phamerator run in Applications phamerator dev phamerator directory Phamerator You will be prompted for username root password MYSQL server password server localhost and database name Ifa popup window appears prompting you to re enter the password you typed incorrectly in the Terminal window Click on the red X and start Phamerator again Start server for processing ClustalW jobs run in Applications phamerator dev phamerator directory pheamoerver InnoDB py U roct p localhost n Localhost d testphage 1 1 L True a clustalw Start client for processing ClustalW jobs run in Applications phamerator dev pha
35. rogress window will automatically disappear and Terminal will display clustalw or blast processing is complete Also the Terminal windows running BLAST or ClustalW will say No work units available Sleeping 12 Assign phamilies phams for gene products in the Phamerator database a Assign phamilies using the data that ClustalW and BLAST have computed Type the following command in Terminal from the phamerator directory and press Enter phamBuilder4 py u root p s localhost d testphage c 0 325 b le 50 b The default parameters for phamBuilder are 32 5 identity and an E value of 1e 50 Phamilies are created when a gene product has more than 32 5 identity or an E value of 1e 50 with another gene product c When this process is complete your Phamerator phage database is ready to use d Ifyou haven t assigned clusters yet you can do that now see V 7 Databases are still functional even if no clusters are assigned 13 Setting up a local copy of the Conserved Domain Database CDD If the Conserved Domain Database CDD has not been set up on your computer you will need to do the following steps They are also rather time consuming and need lots of processing power but you only have to do it once Make sure that you have at least 40 GB free on your Ubuntu virtual hard drive a Goto NCBI s CDD website ftp ftp ncbi nih gov pub mmdb cdd b Download the file cdd tar gz this needs to be don
36. sql server password the server address localhost and the database name as prompted by Terminal If a popup window appears asking you for your password again you typed it wrong Click the red X and start Phamerator again After Phamerator starts click on the Phages tab on the left If all of the information is correct move the GenBank file from importseq to either the worked or failed folder Move another GenBank file into the importseq folder and go to the Terminal window By pressing the up arrow on your keyboard the previous command you used is filled in and you can simply press Enter to import the next file Deleting phage genomes from a Phamerator database J AA k You can use the phamerator manage db py script to delete phage entries from the database that did not import correctly You will have to start Phamerator to make sure it was successfully removed phamerator manage db py u root p s localhost d testphage remove name TA17A Substitute testphage for your database name and TAI7A for the name of the phage you are deleting Type it exactly as it appeared in the Phages tab in Phamerator WARNING When you remove phages a message will tell you that doing so invalidates pham assignments and prompts you to either erase them yes or leave them alone no If you want to preserve the existing pham numbers always choose no You can re ru
37. ss of ClustalW or BLAST Assign phamilies phams for gene products in the Phamerator database Setting up a local copy of the Conserved Domain Database CDD Using the Conserved Domain Database CDD Search Modifying the Phamerator Database HOW TO USE THE PHAMERATOR USER INTERFACE TO EXPLORE THE DATABASE Displaying linear genome maps Displaying phamily circles Exporting Pham and Cluster Tables Exporting genomes genes or proteomes Accessing the Phamerator Quick Start User Manual CONCLUSION TROUBLESHOOTING AND FREQUENTLY ASKED QUESTIONS APPENDIX OF PHAMERATOR TERMINAL COMMANDS 134 I INTRODUCTION Computer software is used compare bacteriophage genomes Phamerator was written by Dr Steven Cresawn to analyze Mycobacteriophages that were isolated and sequenced as part of the SEA PHAGES project headed by Dr Graham Hatfull Phamerator is currently being used to analyze many other kinds of phages Phamerator is compilation of Python scripts that runs in Linux Ubuntu Different scripts import GenBank formatted phage genomes into an SQL database perform BLAST and ClustalW computations group similar gene products into phamilies and identify conserved domains in gene products Each of these scripts records the results in the SQL database The user interface reads the database displays information and has many forms of data output including custom sets of genomes proteomes linear genome maps phamily circles and tables BLAST Clu
38. stalW create families identify conserved domains Import phage senom s Phamerator GenBank files Database Data output genome maps pham circles pham tables Phamerator User Interface To install Phamerator and set up the database users need to use Terminal the Ubuntu command line interface called Terminal These instructions will describe how to set up Ubuntu install Phamerator and the programs Phamerator needs in order to run set up the SQL database import prepared phage GenBank files sequences and perform computations on the database and use the Phamerator graphical user interface to explore your database Using Your Own Databases in Phamerator Setting Up Ubuntu Installing Creating Phamerator Databases 135 Il HARDWARE REQUIREMENTS These instructions will work for a computer running Windows Mac OS X or a computer running Ubuntu natively There are several requirements to setting up Phamerator To explore Phamerator databases you will need a computer that has at least 20 GB free on the hard drive has at least 4 GB of RAM and has at least a dual core processor Building Phamerator databases requires significantly more hard drive space and computing power We recommend a computer with a 64 bit operating system an 17 processor or equivalent and at least 8 GB of RAM The time needed to process a database increases exponentially with each phage added to it If adding conserved domai
39. t get install nebi bilast sudo apt get install bazaar sudo apt get install python pygoocanvas sudo apt get install python webkit sudo apt get install python mysgqldb sudo apt get install mysgl server Sudo apt get anstalhl python pxp sudo pip install numpy sudo pip install biopython sudo apt get install python biopython sudo apt get install python pE sudo apt get install blast2 Install Phamerator run in Applications directory git clone https github com byuphamerator phamerator dev git Install Phamerator run in Applications phamerator dev directory git pull Start Phamerator run in Applications phamerator dev phamerator directory gt Phamerator Create new MYSQL database can be done in any directory mysql u root p e create database testphage Prepare MYSQL database for Phamerator use run in Applications phamerator dev phamerator sql directory Mysql U Foot p Pestphage db seneme sqi Delete MSQL database can be done in any directory mysql u root p press Enter type the password and press Enter drop database testphage and press Enter Export MYSQL database to single file run this command in the directory where you want the file to end up mysqldump u root p testphage gt testphage sgql Compress SQL file Right click on SQL file and choose Compress Import saved SQL file into new database Create new MYSQL database P
40. type the following command as it appears below Then press Enter This command will download Phamerator from a website and put a folder called phamerator dev in your Applications folder git clone https github com byuphamerator phamerator dev git Change into the new phamerator dev folder cd phamerator dev and type the following command Then press Enter git pull Each time you prepare to run Phamerator you should download the latest updates of Phamerator by navigating to the phamerator dev folder and running the git pull command 2 Start Phamerator and download BLAST ao Oo PS After running a git pull typecd phamerator to navigate to the phamerator folder Type Phamerator and press enter through all of the prompts if there are no databases to load Phamerator will start running and ask you if you want to download BLAST Allow it to do this Congratulations Phamerator is now ready to use 139 e Torun Phamerator for the username type root for the password use the same password you used when you installed MYSQL Server for the server use Localhost if running a database on your own computer and for database use the name of the database you want to load IMPORTANT NOTE In command line capital letters and lowercase letters are understood to be different If something doesn t work check to see if you have capital and lowercase letters in the correct places vV HOW TO BUILD A PHA
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