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Omixon Target User Manual

1. Y VY VY w E Programming E Sound amp Video gt O system Tools gt By clicking on this icon the Omixon Target application starts and is ready for use If you want to uninstall Omixon Target an uninstaller is available in the directory where the application was originally installed The uninstaller shows the following two dialogs Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Omixon Target Application Uninstall Are you sure you want to completely remove Omixon Target Application and all of its components Click Next to continue or Cancel to exit Setup h Cancel In the next step we are asked about Do you want to remove your working data By default the checkbox is not selected and the directory ogve which is the data directory for Omixon Target will not be deleted If you intend to use future version of Omixon Target and you don t want to be deleted all your work visible in the Omixon Target then just skip this step Instead if you want to cleanup the working directory then selet the option Yes Omixon Target 0 1 Uninstall Select Additional Tasks Which additional tasks should be performed Select the additional tasks you would like Setup to perform while uninstalling Omixon Target then click Next Yes L Do you want to remove your working data Next gt Cancel Copyright 2012 Omixon Biocomputing Kft O
2. Setup Omixon Target Application 1 0 0 Installing Please wait while Setup installs Omixon Target Application on your computer Extracting files GMT 14 _ _ _ _ _ _ _ ce_ ee install4j At the end of the installation process the following dialog indicates the success of the installation Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Setup Omixon Target Application 1 0 0 Lo ae Completing the Omixon Target Application Setup Wizard Setup has finished installing Omixon Target Application on your computer The application may be launched by selecting the installed icons Click Finish to exit Setup After the installation the following icon becomes available in the Start menu _ WHLIYVOVUIL WIIG Wruru cvus Ml gt Omixon Target ad Nero Express LS Paint gt All Programs Search programs and files p The Omixon Target application can be started by clicking on this startup icon Mac OS X installation guide As Mac OS X is an exclusively 64 bit operating system and it always has a proper Java Runtime environment for our tool therefore the installer does not contains Java Runtime Environment The installer is in DMG format In Finder file manager the installer file should look like this Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Downloads
3. e Choose one or more sequencers for your Analysis tip use CTRL or SHIFT click to select multiple items Press Next e Select Annotation Type Amplicon Press Next e Select the AmpliSeqCancerAmplicons bed file from the file system e Leave the next step set to Download file s e Press Finish This will start a new background task This task will take quite a lot of time it will download most of the HG19 genome and install this reference data into the tool The task will parse the bed file and identify which chromosomes it needs from within the bed file It is important that the bed file uses the standard HG19 notation for chromosome names i e the name has to be chr1 and not just 1 If the chromosome is already configured within the tool then it will not be downloaded again Once this task has completed you should see your new Experiment and Analysis and you can start importing sequencing data and using your new Target Download problems If for some reason you cannot download files from within the tool e g due to network security settings you can download the files manually and use the Select Local file s option instead of the Download file s option in the wizard The pre packaged HG10 zip files can be downloaded from here e http omixon download s3 amazonaws com target_ref_hg19_chr1 zip e http omixon download s3 amazonaws com target_ref_hg19_chr2 zip etc replace the chr number with the chromosome y
4. HS Y 5 gt Bi omixon ta 1_0_0 dmg FAVORITES Dropbox E All My Files AirDrop A Applications xa Desktop a Documents Downloads Movies Once the DMG archive is opened you can see the installer icon E Downloads omixon target Omixon Target Application Installer This has to be started like any other Mac OS X installers Note that if you don t already have Java installed on your Mac it will automatically be downloaded and installed at this point Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Application Setup Wizard This will install Omixon Target Application on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup cancel The next step is to set the destination directory Select Destination Directory Where should Omixon Target Application be installed Select the folder where you would like Omixon Target Application to be installed then click Next Destination directory Applications Browse Required disk space 55 5 MB Free disk space 59 453 MB install4j lt Back Next gt Cancel The default destination directory on Mac OS X is Applications By clicking on the Next button the installer performs the installation process and the final dialog will look like the following Copyright 2012 Omixon Biocomputi
5. Xmx5g Changing this to Xmx69 saving the file and restarting Omixon Target will make 6GB of memory available instead of the standard 5GB Run the tool with reduced sensitivity If you are running with a 32 bit version and or you don t have more than 5GB of memory available the only other option is to reduce the sensitivity of the mapping tool This is done by using the Advanced Parameters tab of the Map and Align wizards In here you can select a file You need to create a txt file called something like advanced properties the exact file name doesn t actually matter and add the following line to the text file orm sampling default 4 This will cause the mapping tool to sample every 4th position in the reference rather than every single position This saves a lot of memory 60 to 70 of the total You can also of course try a smaller value such as orm sampling default 2 This will save about 30 40 of the memory required Dashboards Most of major functions of Omixon Target are organised into separate Dashboards The section provides an overview of the Dashboards and the concepts behind each of these main functions Home Dashboard This is main home page for Omixon Target There are only three functions available from this screen e Data analysis e HLA Typing e Settings The Data Analysis function is for general analysis of sequencing targets where sequencing data can be mapped and aligned against a referenc
6. Once all these properties are set for an Analysis there will be automatically used by all the wizards and processes for the Samples that belong to the Analysis Design Experiment This wizard usually only has to be run once when a new Experiment is created This wizard inherits all its settings from the Profiles have been added to the tool and the lists of configured Reference Genomes and Targets The steps in the wizard General Properties Choose Sequencer Choose Target Choose Species Choose Genome Choose Contig Once all these properties are set for an Experiment there will be automatically used by Analyses that belong to the Experiment Create Reference This wizard is used to create a new Reference Genome within Omixon Target Once created this Reference will become available for all Experiments and Analyses to use The options in this wizard e Choose Reference Name e Select Species It is not sufficient to simply create a Reference Genome before it can be used with in the tool it should also have reference data known variants and gene exon annotations imported Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Import Reference Data This function will import the reference data for the Reference Genome It is recommended to import and use whole chromosomes Even targeted sequencing can place some reads off target and it s very important that off target reads are also mapped off target
7. i e this will delete all imported and analysed data and reset the whole of Omixon Target back to a brand new status as well as set up the application again Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual only with the Expert Profile This function is essentially only recommended for demonstrations or if you really do want to start again from scratch Data Analysis Dashboard The Data Analysis Dashboard is the starting point for general genomic analysis tasks The recommended way to start doing data analysis is to use one of the in built Profiles The tool starts with an empty Expert Profile The first step is either to manually configure this Profile to add one of the existing pre configured Profiles to the tool or to create your own Profile s There are three pre configured profiles available e BRCA e HLA e CFTR The pre configured Profile to be used can be chosen with the Add Profile function This will start a new background task which will download and import all the data required to configure the tool for the chosen profile In order to create a custom profile the Create HG19 Profile or the Create Custom Profile functions can be used With the Create HG19 Profile only a single annotation file in bed or gff format is needed in order to configure the profile the reference data and the known variants can be downloaded and imported automatically The Create Custom Profile makes it e
8. empty cells Note that allele candidates with a lower coverage than the selected limit are NOT shown on the HLA Typing allele result dashboard which contains the detailed results For each allele one or more allele candidates can be assigned There are four different ways to assign allele candidates 1 Candidates can be assigned manually by clicking on the checkmark before the allele candidate s name 2 All Winner candidates i e the best allele candidates based on coverage statistics can be assigned using the Assign Winners button 3 All unambiguous results can be assigned using the Assign Unambiguous button 4 All allele candidates can be assigned using the Assign All button Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual All assignments can be deleted by the Unassign All button The results can be exported in TXT tab delimited text CSV comma separated text or XLS Excel format Tip If you would like to export only the assigned allele candidates click the Assigned Only button then export the results By clicking on the Browse results button the HLA genome browser can be opened This HLA visualisation tool shows the short reads in the Sample aligned to the allele candidates The best allele candidate is marked as Winner HLA Genome Browser The HLA Genome Browser allows visual inspection of genomics data Multiple allele candidates can be browsed together The data items that can be vis
9. forward forward Import Mapped Data The first goal of Sample analysis is to identify some variants There are three ways to do this and this function allows one of these three ways If you have short read data that you have already mapped from another source you can skip the Import Sequencing Data and Map and Align functions altogether and just import your mapped data into the tool in either sam or bam format and then use the Call Variants function to identify the variants This wizard also allows you to import Sanger data however the Sanger visualisation is currently under development Steps in this wizard e Select Mapping Type e Select Input File e Select Reference Contigs After you have imported mapped data you can browse it via the Genome Browser or use the Call Variants function to identify variants within the mapped data Import Variant Calls The first goal of Sample analysis is to identify some variants There are three ways to do this and this function allows one of these three ways If you have already mapped your data and called variants on it or if you have variants already identified from another source such as Sanger sequencing then you can skip the Map and Align and Call Variants functions altogether and simply import your variants into the tool This function also allows you to import expected variants If you are working with simulated data or if you already have known variants from anoth
10. you can select multiple files from the list in one go e g chr1 fa chr2 fa This will start a new background task Once this task has completed you can proceed to the next step Importing Known Variants This is an optional step but having known variants within the tool can assist with the Variant Calling algorithms If you are still in the Reference Genome Dashboard then you are starting in the right place Otherwise start at the Data Analysis Dashboard click on References and then click on the new custom reference in the list e Select the Import known variants item in the Actions menu on the left Select the matching vcf files for the chromosomes Note that we need the name of the chromosome e g chr1 to appear in the name of the matching vcf file here is an example of one we use within one fo the Profiles dbsnp hg19 chr1 vcf This will start a new background task Once this task has completed you can proceed to the next step Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Import Target You should start at the Data Analysis Dashboard e Click on Targets and choose the Import Target button from the menu above the list of Targets e Give your new Target a name e g Custom Cancer Gene Panel e Select the Annotation Type Amplicon e Select the bed file for your target regions from the file system e Choose the Reference your new custom reference e Select the Contigs chromosomes e Press Fi
11. Hye Ghee Id Sole co 38 1519 Design ANa VSS A e ads id deplete dns 38 125 20 Design EXPO cin ace asec Ai Pages een dee ater AS Wana ne aI aan eso neh ea alert 38 1 5 21 Create Reference ir oteriana pae bes A A pee eee a AEE 38 1 5 22 Import Reference Data ici ete cask is pea ea here ee rds Oe a bend when a det eee de ce 39 125 23 Import Known Variants cris ee Sebo is A eS eka res die Baad Poe A Bb oe eee eS Ure cda 39 15 24 MPORsANMOTALONS 2 dd a ais 39 1 35 25 Create TarQet amni A a Mag Se dean ab eked Bed gee one 39 1 5 26 Configure Target ie ioten insde kep ord ee beta Satan ChdG anche eas Rhee eee eae 39 15 27 Import Target Annotations ae nea eaa era Araia td a Bye dale A e dae 39 15 28 Export Mapped Data ici A A A lr Peas A A e rake A 39 1 5 29 Export Mapped Regions wsixc cence cee eee ee eee ye yuk See eee a ee pull ele ee ee te eo tO 40 1 5 30 Export Approved Valiants rae cian Ra a o tc py ada A al da 40 1 25 31 Export Activity LOg cas seus ilk Raga ae dieses Wie veil beac Seca EO Bea eh ee let A A lath Bates PAE 40 1 5 32 Reset Everything 22 ct eve cee tae cee a eee 40 VIOAPPOAADE prin oes ti A A Meme we ae avenue wh ah Wate ware a Nob aoe AAA ic 40 1 61 Keyboard Shortcuts coords heed ners Eo bel wpe BY OR RARE ooh Eire ta ad a tabs re eA CoS 40 Omixon Target User Manual Omixon Target User Manual Introduction Introduction Omixon Target the tool is a suite of software for analysing targeted sequenc
12. can import it with this function and then run the Map and Align function which will align the data to a Reference Genome and run a variant call in order to identify the variants You will usually either run this wizard from within an existing Sample or use the Import Multiple Samples wizard from the Analysis Dashboard which both imports data and creates multiple samples at the same time This function will allow you to import two FASTQ files at the same time or one SFF The second one is optional and is for paired reads if your analysis produced paired end or mate pair data If using paired reads the two input files are assumed to have the exact matching reads in the exact same order there is currently no in built check for this The SFF import doesn t support paired mode and is only available in Omixon Target Pro Steps in this wizard e Select Input File e Select Paired Read Options After you have imported sequencing data you can align it and call variant in the data using the Map and Align function Paired Read Options e Minimum Distance the minimum expected distance between the two reads e Maximum Distance the maximum expected distance between the two reads e Orientation the orientation of the reads with respect to each other The first file is the first in the orientation as well Options are FR forward reverse i e first file contains forward reads second file contains reverse reads RF reverse forward and FF
13. default setting is 20 Settings selected in the Configure Application wizard are saved and will still be in effect after Omixon Target is restarted Wizards Wizards are dialogs that pop up on top of the main display area in Omixon Target and allow the user to choose parameters and options for various processes In all cases as many options as possible are already prefilled and preselected within the wizards If only one Target one Sequencer one Reference Genome etc are defined then there will be no need to select these again within the wizard In many cases there will not be any need to change any options within the wizards at all they can be started and the Finish button hit with no other interaction required Upload license You can upload a license with this wizard Just select the file you ve got from Omixon and finish the wizard The license can be an upgrade from an evaluation license to a full license either permanent license or annually renewable or could just be a top up license for the credit based services such as HLA Typing HLA Typing Setup This only needs to be run once unless you would like to use another sequencer e Choose the sequencer s you would like to use and hit finish Tip you can use CTRL and SHIFT clicks to select multiple sequencers A configuration file will be downloaded from the internet and installed into Omixon Target This should only take a few seconds Once this has finished yo
14. directory is home user omixontarget This is the most simple approach to install in various linux distributions The next step is symlink location selection Setup Omixon Target Application 1 0 0 Select Directory for Symlinks Where should Omixon Target Application create symlinks to the executables Select the folder where you would like Omixon Target Application to create symlinks then click Next Y Create symlinks Destination directory usr local bin Browse k lt Back Next gt Cancel The default location for symlink is usr local bin but one may choose any other directory This symlink is useful for users who usually start the application from the command line Since the installer is creating a startup icon in the system menu this symlink is optional for most users Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual After clicking the Next button the installation process starts After it is finished the final screen appears Setup Omixon Target Application 1 0 0 Completing the Omixon Target Application Setup Wizard Setup has finished installing Omixon Target Application on your computer The application may be launched by executing the installed start scripts Click Finish to exit Setup Once the application is installed an application icon can be found in the System menu es Places System a I ws Accessories A Graphics internet
15. on 3 platforms e Mac OS X e Linux e Windows The recommended hardware requirements for all the optional modules are the following m 64 bit multi core CPU with 64 bit operating system atleast 8GB memory storage space requirements mostly depend on the size of data usually used for analyses The memory requirements depend on the size of the reference genomes being used The memory limitation of 32 bit operating systems is the primary restriction for not fully supporting these For the Genome Browser Module only or for demonstration purposes with small genomes the tool can still be used on a 32 bit operating system with up to 4GB memory An Oracle Java 6 Runtime Environment JRE is required this is always included inside the installer Installation guide There are separate installation packages for all three supported operating systems In the following sections are descriptions for the installation steps required for each operating system The Omixon Target Pro version of the product has also a separate section to summarize installation and setup instructions related to that specific type of distribution Windows installation guide Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual There are many versions of Windows operating systems We have tested Omixon Target with Windows 7 Windows XP and Windows 8 We provide two versions of the installer package for Windows system both are bundled with a Java Run
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17. this with an example In this example we will create the profile for one of the ready to use lon AmpliSeq panels Here s the link to the kit http Awww invitrogen com 1 1 12092 ion ampliseg cancer panel html Here is a description of a run of the kit with some test data if you would like to try it http lifetech it hosted jivesoftware com docs DOC 2180 The amplicons in the kit are documented in a bed file http omixon download s3 amazonaws com AmpliSegCancerAmplicons bed Option 1 Using the Create HG19 Profile function recommended The fastest way to create a new Profile is to use the one of the two Create Profile functions from the main Data Analysis dashboard These wizards wrap up the a number of the other steps explained below There are two options for Create Profile creating one based on the HG19 reference genome or creating a custom one with another reference genome For the AmpliSeq example you can do this very simply by using the Create HG19 profile function This will automatically download and extract all the required reference chromosome data from the Omixon web site You should start at the Data Analysis Dashboard e Click on Create HG19 Profile e Fill in the details Profile Name e g AmpliSeq Target Name e g AmpliSeq Amplicons Experiment Name e g All my Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual AmpliSeq data Analysis Name e g First go Press Next
18. BIOCOMPUTING Y SOLUTIONS Omixon larget Designed with diagnostics in mind IS Omixon Target User Manual 1 Omixon Target User Manual 2s 0ceeseni esa dly creat be a eta pe nga a 4 TARINTOUCION rr tala a a loa 4 US A A A A A A A A A E IRE 4 1 1 2 Sequencing Technologies ooococcoc o 4 TAS Modules saie a A A dada ddan dyer tetany 4 11 4 Key GOnCOpts cuota e Ad Ee are ened 5 4 5 Bioinformatics NOTES aaa o e tn ali A RA iR IR 7 1 1 6 System requirements 0 ii a a A da db 7 141 7 Installation guide mc a ee beatae ats 7 1 1 7 1 Windows installation guide 2 0 1 icc ce eee tee ee ee ee eee tent ence renee 7 1 1 7 2 MacOS X installation Quide s aa ot occurs gees tt e daa Eee oe 10 1A A SsLINUe installation guide F suecia iat aer taa O O A aaa Aina 13 1 1 7 4 Pro Data Analysis Module installation notes ooocoococcccocor oc 18 1 1 8 Acknowledgements 20 ovocroncr A ee Eea aA 19 1 2 TUOMAS Y is A ad WS fue te Daa id hsb a stance fi ad il dias 19 12 EUSHUSC Scares bsp wkd rpa eed wegen E a eines ria Eira E Hie ETES E bedrest ote beater 19 15213 0 A0dINga Profils ar ao lll ata eae a eaaa a A aval loci 20 1 2 1 2 Import sequencing fastq data 2 eee eee 20 1 2 1 3 Map the reads and call variants 0 0 eee eee 21 1 2 1 4 Results analysis and visualisation 2 1 0 0 0 eee eee 21 12 2 Custom Profiles vecinita mr abba ord 21 1 2 3 New Target iceciedeue levis t peed eee SE Ree Tee ee a ele Cad pulell
19. Experiment configuration Advanced Options e Parameters file You can choose to import a properties file with some advanced parameters This file is the same one used for the Omixon Variant Toolkit the command line version of our alignment algorithms you can find the latest readme file for this linked via the Omixon web site the readme file link is in the Useful Links section at the bottom of the page https www omixon com omixon abouttoolkit htm Maximum Coverage You can choose what the maximum depth of coverage should be for your results For very deep coverage data it s usually enough to keep 1000 deep short reads however if you want to you can keep more or less than this This will cause your actual mapping results to be discarded only a number matching your maximum coverage will be kept Variant Call Options You can choose whether or not to run a GATK variant call after the map and align function has run You can always run the variant call later if desired The dbSNP known variants file is an optional parameter You can either use dbSNP data that has been imported into the tool or select a dbSNP file from the file system Import Sequencing Data The first goal of Sample analysis is to identify some variants There are three ways to do this and this function allows one of these three ways Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual If you have short read data from sequencing run you
20. GFF format using the Import Target function Clicking on a Target will take you to the Target Dashboard where the Target can be configured Experiment References Configure References for an Experiment Omixon Target has a centralised configuration for the References reference sequences used in the Data Analysis portion of the tool These References can be set up once and then used in multiple Experiments and Analyses This screen lists the Reference Genomes linked to this Experiment and allows Reference Genomes to be created or deleted Clicking on a Reference Genome will take you to the Reference Genome Dashboard where reference data in fasta format can be imported along with known variants from dbsnp Experiment Targets Configure Targets for an Experiment Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Omixon Target has a centralised configuration for the Targets essentially lists of annotations used in the Data Analysis portion of the tool These Targets can be set up once and then used in multiple Experiments and Analyses This screen lists the Targets attached to this Experiment and allows Targets to be created or deleted It is possible to create a Target by importing annotations in BED or GFF format using the Import Target function Clicking on a Target will take you to the Target Dashboard where the Target can be configured Analysis References Configure References for an Anal
21. Kft Omixon Target User Manual e Select the Sample you wish to work with by clicking on it moves to the Sample Dashboard Select the Import sequencing data button and choose a fastq file or a pair of files for paired data from the file system Click Finish This will start a background task Once this task is finished move to the next step of the tutorial Map the reads and call variants Once you have some fastq data imported then you can map and align this data plus call variants in a single function Either Select a Sample with fastq data and from the Sample Dashboard select Map and Align from the buttons on the left or e Select a Sample or more than one from the list of Samples in the Analysis Dashboard and select Map and Align from the buttons on the top e This will start the Map and Align wizard You shouldn t need to change any settings in here you can review the setting and when ready press Finish This will start one background task per Sample Once the background tasks are finished go to the next step Results analysis and visualisation Once the Map and Align including variant call is finished you will now be able to start analysing the results You should see two new data items within the Sample one for the mapped data in BAM format and one for the variant calls in VCF format You can visualise the results in the Genome Browser From the Sample Dashboard select Genome browser or from the Analys
22. Manual exported again either in sam or bam format The export will also create a standard index file for the exported data Export Mapped Regions From the Genome Browser it is possible export the short read that overlap or are contained within the currently visible region There are two options for the export the reads can be exported in either fastq format for re mapping or in sam bam format Export Approved Variants This function is currently the final step in the data analysis pipeline Once you have a short list of approved variants you can export an annotated vcf file from the tool using this function Only variants that have been approved either by the manual or auto approval functions can be exported in this way Export Activity Log This function will export the activity log into txt csv or xls format The log can be for a single sample a whole analysis a whole experiment or the whole application It is useful for obtaining a history of everything that was done within the tool for that sample for the purposes of documenting an experiment Reset Everything It is possible to Reset everything reset the whole of Omixon Target to an empty state Resetting will delete all imported and analysed data Resetting is only recommended if it s the first thing that is done with the tool if the tool is being used for demonstration purposes or if you really do want to start again completely from scratch
23. Reset Everything is available from the Settings Dashboard You will be asked again if you are completely sure before the reset process is started There is no way to recover data that is removed by a reset Once you have reset the tool will then only contain the Expert Profile The next recommended step is to use Add Profile to add one or more of the pre configured Profiles to the tool which will download and import all the data required for each Profile Appendix Keyboard shortcuts The following generic keyboard shortcuts are available in Omixon Target e F8 key closes the window and exits the application e F11 key switches to fullscreen display mode to utilize as much space as possible for visualization note that certain platforms and window managers are not working properly together with this function the first time you use you will get a warning message about this Copyright 2012 Omixon Biocomputing Kft
24. TRL or SHIFT click to select multiple items Press Next Select Annotation Type Exon Gene Amplicon Press Next Select a bed or gff file from the file system containing the annotations that describe your Target Press Finish Create Profile The tool starts up in Expert mode with only the Expert Profile configured and no reference data imported or targets set up A Profile is simply a wrapper around a Target plus a set of reference data and known variants A Target is simply a list of reference annotations for either genes exons or amplicons It is possible to add a pre configured Profile to the tool using the Add Profile function or you can use this Create Profile option instead It s also possible to skip Profile set up entirely and simply configure the tool manually e Fill in the details Profile Name Target Name Experiment Name Analysis Name A new Profile Target Experiment and Analysis will be created Press Next Choose one or more sequencers for your Analysis tip use CTRL or SHIFT click to select multiple items Press Next Select a reference Press Next Choose the reference files Press Next Optional Step select the matching Known Variants vcf files for the reference files Note that we need the name of the contig e g chr17 to appear in the name of the matching vcf file here is an example of one we use within one of the Profiles dbsnp hg19 chr17 vcf Press Next e Select Annotation Type Exo
25. amples that are sequenced there is a simple data management hierarchy in the tool The sequencing data will belong to a Sample A number of samples can be grouped together within a single Analysis And finally a number of analyses can be grouped together within a single Experiment The configuration of shared data such as the target regions for the investigation e which reference species genomes will be used and the imported reference sequence data and known variations and the sequencer used for the sequencing of the data can be done at both Experiment and Analysis level Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual If desired a more generic configuration can be created within an Experiment with a more specific configuration chosen within each Analysis For example it might be desirable to separate the Analyses by sequencer if multiple sequencers are being used It s also possible to use a sub set of targets within the Analysis for example the HLA A gene could be the target of one Analysis and the HLA B gene could be the target of another There is an Experiment Dashboard an Analysis Dashboard and a Sample Dashboard to manage each of these Profiles The easiest way to use Omixon Target for data analysis is to use one of the pre built pre configured Profiles This already contains a Target definition and all the reference data required for a particular Target Adding a new profile will c
26. arked as Winner By clicking on the Analyse button next to the allele name the HLA Typing allele result screen is opened This page contains detailed exon level statistics for the allele candidates A minimum coverage threshold can be set for the summary result table This limit can be set as an absolute coverage value i e the mean number of reads for the allele or a relative coverage threshold minimum coverage relative to the top allele The default value for minimum coverage is 10 for the absolute and 95 for the relative coverage filter function Alleles below the coverage limit are shown as empty cells Note that allele candidates with a lower coverage than the selected limit are NOT shown on the HLA Typing sample result dashboard which contains the detailed results For each allele one or more allele candidates can be assigned There are four different ways to assign allele candidates 1 Candidates can be assigned manually by clicking on the checkmark before the allele candidate s name 2 All winner candidates i e the best allele candidates based on coverage statistics can be assigned using the Assign Winners button 3 All unambiguous results can be assigned using the Assign Unambiguous button 4 All allele candidates can be assigned using the Assign All button All assignments can be deleted by the Unassign All button Note that the Assign buttons on this screen affect all the displayed samples if you prefer to d
27. asy to create a profile with a custom e g HG18 or non human reference as it merges three of the other functions within the tool into a single wizard for convenience Import Reference Data Import Known Variants and Import Target Once a Profile has been added or created you can get started by selecting one of the Experiments listed in this Dashboard The targets and references for the whole tool can also be configured from here Experiment Dashboard The Experiment is the top level of the small hierarchy organising the genomic data within the tool This is a flexible container and should be used to reflect the nature of your analysis requirements An Experiment is a grouping of Analyses Common configuration elements for a group of Analyses can be specified at the Experiment level including the references to use the sequencer and the targets This will restrict the Analysis configuration options and later the options available to the various analysis tasks with the tool The Experiment can also be used for analysing and reporting the results of multiple Analyses together Analysis Dashboard The main purpose behind the Analysis is to group together a set of samples These Samples will share common configuration within the tool they will share the same targets the same references the same sequencer etc The Analysis inherits its configuration from its parent Experiment and can be used to help split the Exper
28. ause the data to be downloaded and automatically configured with the tool It will also create and configure a starting Experiment Analysis and Sample plus import some example Sample data if required Reference Genomes One or more reference genomes can be configured for each Experiment One or more contigs usually chromosomes can be chosen for each reference genome The reference genomes and or contigs can split across the Analyses The sequence data for the reference genome fasta format will need to be imported into the tool For the best variant analysis results the dbSNP known mutation data for the reference if available can also be imported into the tool There is a Reference Genome Dashboard to manage the data for the reference genomes Samples Each Sample will generally consist of the sequencing data or results for a single individual There are three possibilities for importing sample data e The raw sequencing data short reads can be imported into the tool in fastq format after which it can be mapped and aligned against one of the reference genomes one or more contigs and variants can be called detected all in a single step e Itis also possible to import short reads that have been mapped by another tool and just do the variant detection or simply just browse the data in the Genome Browser Finally variant call results can be imported alone without any short read data at all and visualised and anal
29. correctly particularly if they happen to map to a pseudogene of the gene s being targeted The input file must be in fasta format We recommend using multiple fasta files within Omixon Target rather than Multifasta references Contigs will be automatically created based on the names found within the input fasta file Import Known Variants This function will import doSNP known variant annotations for the Reference Genome This will be used later with the GATK variant call If the Reference Genome has been configured to use a whole chromosome then we recommend importing all matching dbSNP records for that chromosome The annotations must be in vcf format Import Annotations This function will import Gene or Exon annotations for the Reference Genome This will be used later for Target configuration Currently the annotations must be in bed or gff format If you need another format please let us know The steps in this wizard are e Select Annotation Type e Select Input Files e Select Contig Create Target The wizard will allow the creation of a new Target The other ways to create a target are to use the Import Target wizard and import annotations plus create a Target in a single step or the Create Profile wizard which merges the Import Reference Data Import Known Variants and Import Target wizards into one A Target is defined as a list of annotations These annotations can be for genes exons or am
30. d E Target configuration via Targets lists and Target Dashboard e Create Target P E Adjusting Memory Usage Import Annotations Genes or Exons e Configure Target When you install Omixon Target it comes preconfigured with some memory settings These will vary depending on which version of Omixon Target you have chosen to install For the 64 bit versions the memory is set to 5GB for the 32 bit versions it is set to 1200MB For most analysis the maximum 5GB memory will be enough However it can happen that this is not sufficient memory for the underlying alignment algorithms to run If you get Out of Memory or GC Limit Exceeded errors then you will need to either e increase the amount of memory available to Omixon Target or run the tool with reduced sensitivity The first option will be preferable Running with reduced sensitivity is possible and saves lots of memory but doesn t give such good results Increase the amount of memory available to Omixon Target This can be done by editing one of the Omixon Target configuration files In your Omixon Target installation directory on Windows and Linux you should be able to find a file called ot gui vmoptions and on the Mac it is found in Applications OmixonTarget app Contents Info plist If you edit this file you should find a single VM virtual machine option which looks like this Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual
31. d Quality Control steps are optional The Variant Call can be performed later using the Call Variants function or the mapped data can be copied elsewhere and other variant caller used instead The Quality Control cannot be run separately only as part of the Map and Align process The steps in this wizard Select Genomic Data Select Species Select Sequencer Advanced Options Variant Call Options Quality Control Options coming soon Like all the wizards within the tool most of the options within this wizard will already be pre filled and pre selected based on your chosen Analysis and Experiment configuration Advanced Options e Parameters file You can choose to import a properties file with some advanced parameters This file is the same one used for the Omixon Variant Toolkit the command line version of our alignment algorithms you can find the latest readme file for this linked via the Omixon web site the readme file link is in the Useful Links section at the bottom of the page https www omixon com omixon abouttoolkit htm e Maximum Coverage You can choose what the maximum depth of coverage should be for your results For very deep coverage data it s usually enough to keep 1000 deep short reads however if you want to you can keep more or less than this This will cause your actual mapping results to be discarded only a number matching your maximum coverage will be kept Variant Call Options You can choose whether or
32. e Custom and manually set how many reads to process If you are not sure then use the All option to process all the reads For very targeted data sets including only the HLA loci this value doesn t need to be too high about 10 000 For larger kits such as the RainDance HLAseq kit the whole MHC this will need to be significantly higher at least 2 000 000 For whole exome or whole genome data sets it is recommended to process all the reads or pairs because of the lower coverage that is usually found in these kind of data sets Add Profile The tool starts up in Expert mode with only the Expert Profile configured and no reference data imported or targets set up A Profile is simply a wrapper around a Target plus a set of reference data and known variants It is possible to add a pre configured Profile to the tool using the Add Profile function The available pre configured profiles are e HLA chr6 BRCA chr13 and chr17 e CFTR chr7 The three pre configured profiles can be set up automatically by using this function which will also download and import all the data required for each Profile You can choose not to add a pre configured Profile and create your own using the Create Profile option It s also possible to skip Profile set up entirely and simply configure the tool manually Download problems If for some reason you cannot download the profile configuration file from within the tool e g due to network secu
33. e of installers are the most suitable to install on various Linux distributions After you get the installer file the shell script still does not have the permissions to run directly You need to open a terminal window to Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual make the installer executable with the following command chmod x omixon target_unix_1_0_0_with_jre sh Immediately after that the installer can be started with the following command omixon target_unix_1_0_0_with_jre sh Once the installer is started the following screen appears Setup Omixon Target Application 1 0 0 Welcome to the Omixon Target Application Setup Wizard This will install Omixon Target Application on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup By clicking on Next button the next step will be the destination directory selection Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Setup Omixon Target Application 1 0 0 Select Destination Directory Where should Omixon Target Application be installed Select the folder where you would like Omixon Target Application to be installed then click Next Destination directory home tibor omixontarget Browse Required disk space 144 5 MB Free disk space 89 376 MB k lt Back Next gt Cancel The default destination
34. e of them may be off target and therefore not interesting Some may only have very low coverage and so are not trustworthy Some may be complex and require manual inspection to figure out what is actually going on in the underlying short read data Variants can be approved or rejected in the Approval Dashboard All variants start in pending status and there are functions to automatically approve and reject groups of variants by various criteria and also to manually approve or reject individual variants as well Downstream analysis steps will only consider approved variants for their input Reference Genome Dashboard This is where the data for the Reference Genomes is managed In here the actual fasta data for the reference genome or chromosome can be imported Contigs will be automatically created when the fasta file is imported into the tool We strongly recommend that whole chromosomes are used as the reference sequence to allow for any off target reads that might fall on pseudogenes to be properly mapped to the pseudogene and not cause false positives by being incorrectly mapped to a gene Known variants from dbSNP can also be imported in vcf format These are important for the quality of the Variant Call and it s recommended to import them It s also possible to import gene and exon annotations in vcf format These can be used for creating new Targets within the tool If you don t need to create a new Target then you don t ne
35. e sequences and variants can be called visualised and analysed The HLA Typing function is only for determining and visualising the HLA types within a set of NGS sequencing data Itis simple fast and accurate The Settings function allows you to manage users licenses and general settings within the tool plus reset the tool to an empty starting state HLA Typing Dashboard All the HLA Typing functions are available from here Firstly the Setup Typing function should be run This only needs to be run once Once the Setup Typing function has been run then the HLA Typing function can be used From here multiple samples can be scheduled for typing simultaneously It s possible to view the results from the screen as well as to delete results from previous runs Results from multiple analyses can be viewed simultaneously Tip You can use CTRL or SHIFT clicks to select multiple analyses In the header section of the dashboard you can see the version and date of the IMGT HLA database that is currently configured and the sequencers which have been set up within the tool Settings Dashboard Reachable from the Home Dashboard the Settings Dashboard displays an overview of the settings in the tool and allows access to the user management features the visual display settings license settings and the bug report mail settings There is also the dangerous Reset Everything button This will cause the whole Application to be reset
36. e the selected target regions manually inspect the variants and short reads see a consensus sequence and see the coverage of the short reads Multiple samples can be browsed together Analyse Sample Variants It is also possible to import expected variants into the tool There is a function called Analyse Sample which will automatically compare the expected variants with the actual variants found either imported or via the map align call variants functions These expected variants can come from previous analyses for example with Sanger data or they are also useful if the data being analysed is simulated data with known results The Analyse Sample Variants function also allows the results of the Quality Control to be inspected Sample Difference If multiple samples are selected in the Analysis Dashboard they can be compared against each other using the Compare Samples function which opens the Sample Difference screen They can also be browsed together and compared in the Genome Browser Manual Approval Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual This function is only available within one of the optional Data Analysis Modules Standard or Pro It s possible to approve or reject variations within a Sample Approved variations will become candidates for downstream analysis such as HLA typing Rejected variants will be ignored by downstream tools Approval and rejection can be done either by t
37. ed to worry about this function Target Dashboard Targets can be configured within this dashboard Targets are optional within the tool but are highly recommended as there are a number of analysis features that give better results for well defined targets A typical Target could be all the exons within the BRCA1 and BRCA2 genes At the moment the target configuration is restricted to gene exon and amplicon annotations There are two ways to import annotations either via the Import Target method where annotations can be imported and a Target created in a single step or the annotations to be used can be imported via the Import Annotations function in the Reference Genome Dashboard this second method requires a Target to be created or altered manually in order to make use of these annotations Other Screens In addition to the dashboards there are some other function rich screens that are mostly used for visualising and manipulating the results of the analysis tasks Analyse Sample Variants This screen is the starting point for the analysis of the variants found in a Sample If there are known expected variants already found for this sample these can be compared with the actual variants found during the analysis The variants found are listed according to whether they are on or off target and it s possible to select individual variants and jump into the Genome Browser to inspect them The Quality Control r
38. er source then you can import these and tag them as expected variants When you have then identified variants via an import of actual variants or via a Map and Align or Call Variants analysis then you can automatically compare the expected variants with the actual variants using the Analyse Sample Variants screen Steps in this wizard Select Annotation Type Select Input Files Import Options Select Contig After you have imported variant calls you can visualise them in the Genome Browser analyse them within the Analyse Sample Variants screen and compare them with the variant calls of other Samples using the Compare Samples function Import Sanger This function allows you to import multiple Sanger sequences for visualisation alongside your short reads in the Genome Browser Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual This function is currently under development however you should already be able to see multiple traces for the Sanger data Map and Align If you have imported sequencing data then you can map and align this data to a Reference Genome using this function This version of the Map and Align runs with a single sample This function has three analyses built in to it e Map and Align using Omixon mappers Variant Call using GATK optional or Samtools only in Target Pro edition e Quality Control coming soon The Map and Align step will always be performed The Variant Call an
39. esult 2 teoria Sarena eta a cl pa nsf a cd a caca 30 1 4 14 HLA Genome Browser coocococ nnn een e teen ttn eee 31 1 4 15User Management 2c ccsc eee A beet tbe Eee vee A 32 1 4 16 Contigure Application s0c02 ede a hae A A At BO ee aks See es 32 TS WizaidS sevi ES oh hoe Pie Bete hina Ae hee ened Dead ah toed aud Ga phe a Oe Bentler te 32 15 AE PIO AG ICONS ai rat al whip dana a cecal pasha e nels ta AS a CAnak a Teena bes 32 15 2 ALA Typing Setup A oak ee ode bras ade area dnd Beis AA beeen 32 15 3 ALA TYPING pele ei A ee ee Ra el ee Weed A ge Here ae Lee oe AAA 33 oda H 24 6 E tai lada Lo ar e nd RCE ol EA E A DO ed dos 33 1 5 5 Create HG19 Profile ii a bd a ea bt 34 15 6 Create Profile rora a td nt O A ai 34 1 5 7 Import Multiple Samples renn ere ne eet n etn e teen eee 34 15 8 Create Sample ici ea he ee es Bee ee ee A ee be ee eee Seb eee 35 1 5 9 Map and Align Samples 1 0 0 aa A E aaa ene tenn teen tenes 35 1 5 10 ImportsSequencing Data Huy 40 4 ppbvdas Ke e b a ad eet hea ee 35 1 5 11 Import Mapped Data iee cctewa deka cede a ae ne A motes 36 1 5 12 Import Variant Calls esini Sea aed eee anette da ede ia 36 15 13 lmport Sanger i esis ewe eee A ea toni ey eee ee en ee eg eee qe Sad ee eee 36 LOA MAA ANIC AMO as a eel a on de al a tN Sai dl o id atletas 37 1 5 15 Call Variants ita tb ia e 37 1 516 Auto Approve cs niai puenak a A A ele a RA a 37 1 17 AUTO Rejet iii a A di dada 38 125 18 ALO ESO E at A A A a A hd
40. esults are also summarised on this screen It s possible to start the Approval Dashboard from here in order to approve the variants ready for further analysis and reject any low quality or off target variants that should be ignored Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual It s also possible to navigate to the Report Dashboard to see graphs charts and other summary information about the Sample data Sample Difference This is the starting point for multiple sample comparison for example with trio data mother father child This screen is started by selecting multiple Samples within the Analysis Dashboard and then selecting Compare Samples Variants within the comparison table can be selected and then you can jump to that position within the Genome Browser which will also allow you to browse all the samples being viewed in the Samples Difference screen together Genome Browser The Genome Browser allows visual inspection of genomics data Multiple Samples can be browsed together The data items that can be visualised include e Reference Sequence e Gene Annotations e Exon Annotations e Amplicon Annotations And for each Sample e Actual Variant Annotations e Expected Variant Annotations e Short Reads including Coverage By default the display is masked so that only differences between the short reads and the reference sequence are displayed This mask is done in a stranded fash
41. function is only available in OmixonTarget Pro edition Steps in this wizard e Select Input File Select Paired Read Options Demultiplexing Options Omixon Target Pro edition only After you have imported sequencing data you can align it and call variant in the data using the Map and Align function Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Paired Read Options e Minimum Distance the minimum expected distance between the two reads e Maximum Distance the maximum expected distance between the two reads e Orientation the orientation of the reads with respect to each other The first file is the first in the orientation as well Options are FR forward reverse i e first file contains forward reads second file contains reverse reads RF reverse forward and FF forward forward Demultiplexing Options Omixon Target Pro edition only e Sequencer the sequencer used to produce the multiplexed data e Barcode file text file containing barcode names and related sequences to identify the samples in the multiplexed files Note that Illumina demultiplexing is supported only for paired fastq files and Roche 454 demultiplexing is supported only for not paired sff files lon Torrent data demultiplexing is not supported currently Create Sample This wizard allows you to create an empty Sample You need to give it a name which should be unique within the Analysis and select a species Once a Sample
42. g and dragging with the mouse Drill mode can also be used where the region highlighted by a mouse click and drag will be drilled into once the mouse button is released Jumping Around You can jump to a position in the reference using Jump To Position If you have previously selected a feature within the display such as an annotation or short read then you can jump back to that feature at any time by using the small arrow button at the bottom of the display Filtering the Short Reads You can display more short reads by using the collapsed pile up view You can also page around the short reads using the small arrows at the top of the short read track You can set the page size using the up and down arrows and the current page by using the left and right arrows This is useful when you have deep sequencing and want to scroll through a few hundred short reads at a time Filtering the Allele Candidates The list of allele candidates shown in the HLA Genome browser can be filtered by coverage This limit can be set as an absolute coverage value i e the mean number of reads for the allele or a relative coverage threshold minimum coverage relative to the top allele The default value for minimum coverage is 10 for the absolute and 95 for the relative coverage filter function Current coverage filter settings can be seen in the top right corner above the alignments By clicking the Assigned Only button unassigned allele candida
43. he Automatic Approval or Automatic Rejection functions or via a manual approval process within the Approval Dashboard screen Bioinformatics Notes Reference Sequences By default Omixon Target is set up to use HG19 as the main human reference sequence It is however possible to configure the tool to use another reference sequence Alignment Tools These tools are only available in the optional Data Analysis Modules Standard and Pro The main underlying tool for the Map and Align step is the Omixon Variant Toolkit This is also available as a standalone command line tool The Toolkit uses a properties file for it s parameters and the same file can be used to transfer advanced parameters to the underlying Toolkit while running the Map and Align step within Omixon Target The Toolkit has it s own readme file which is also available for download via the toolkit page on the Omixon web site https www omixon com omixon abouttoolkit htm BWA will be added soon to the Pro Module Variant Calling Tools These tools are only available in the optional Data Analysis Modules Standard and Pro Omixon currently use open source third party variant calling tools The only one included in the Standard Module is the GATK variant caller from the Broad Institute Also included in the Pro Module is the Samtools variant caller mpileup which gives better results for lon Torrent and 454 data System requirements Omixon Target is supported
44. he reference using Jump To Position or jump to one of the annotations within the Target using Jump To Target If you have previously selected a feature within the display such as an annotation or short read then you can jump back to that feature at any time by using the small arrow button at the bottom of the display Filtering the Short Reads You can display more short reads by using the collapsed pile up view You can also page around the short reads using the small arrows at the top of the short read track You can set the page size using the up and down arrows and the current page by using the left and right arrows This is useful when you have deep sequencing and want to scroll through a few hundred short reads at a time Filtering the Variants The variants in the Act Actual variants track can also be filtered This makes use of the Approval features within the tool individual Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual variants can be approved or rejected or still be pending awaiting either approval or rejection This filter will display All variants three lines default then Pending circle Approved tick or Rejected cross Track Setup You can configure which tracks you would like to see in the display Exporting Genome Browser Data You can Copy to Clipboard the details of individual selected items in the display You can also Capture a Screensh
45. iment into more specific sections There could be one Analysis for lon Torrent and one for Illumina data for example or one Analysis for one Target e g the HLA A gene and another for another Target e g the HLA B gene From the Analysis Dashboard it is possible to browse through and work with individual Samples It s also possible to select multiple Samples and work with these together either by launching them in the Genome Browser or by comparing them using the Compare Samples button which will launch the Sample Difference screen Sample Dashboard A Sample represents a single set of sequencing data for a single individual The Sample has a small life cycle within the tool e Create Sample e Identify Variants e Approve Variants Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual e Approve Sample e Analyse Approved Sample and Approved Variants The concept is that the raw variants called may not all be of sufficiently high quality for downstream analysis and the tool supports a number of Approval functions in order to approve or reject variations and filter them down a set suitable for further analysis In the case of a HLA Typing analysis then the downstream analysis step will be to determine the HLA Types of the sample Approval Dashboard Not all variants are interesting Not all variants can be trusted Actual variants that have been discovered during the Variant Call can vary in quality Som
46. ing data from next generation sequencing NGS platforms The main vision behind this tool is to help laboratories move towards using NGS data for the analysis of diagnostic targets There is a Quick Start Guide for Omixon Target available on the Omixon website http Awww omixon com web guest omixon targetquickstart htm Omixon Target is for Research Use Only Not for use in diagnostic procedures Uses Analysis of Diagnostic Targets HLA Typing Omixon Target has a simple HLA Typer for NGS data This tool is easy to set up and run and multiple samples can be analysed together It provides both a visual summary and tables of reports with statistical confidence measures for the accuracy of the results Data Analysis Omixon Target includes preconfigured support for analysing some of the most common human diagnostic targets such as HLA CFTR and BRCA however you can also configure the tool to analyse any gene or region of interest The underlying mapping alignment and variant calling algorithms are intended to meet the high precision and analysis quality control requirements of diagnostics labs You can easily move from a high level mutation summary table to inspect the underlying short read data and based on this visual inspection you can manually approve variants The tool helps to identify amplification artefacts and other error sources using a simulation based validation track as an analysis control The tool can be used for multi sa
47. ion so that forward strand reads pink and reverse strand reads yellow can be easily distinguished within the display The Display Strand function turns this on and off The short reads track can be collapsed which gives a summary view of the short reads and does not allow each read to be inspected in detail Individual items annotations short reads within the Genome Browser can be selected and it s possible to browse elsewhere and then jump back to the selected item or copy the details of the selected item to the clipboard Expanding the display At the top left of the tool is a small control that allows an expanded view This actually works for all screens in the tool but is most useful for the Genome Browser Clicking the same icon again will go back to the usual display Rotating the Genome Browser The vertical Genome Browser view is more useful for comparing multiple samples The view can be rotated to use a horizontal display which is a bit more traditional for Genome Browsers and is more useful when browsing a single sample Zooming By default the Genome Browser starts in Drag mode where the mouse wheel or and keys can be used to zoom in and out and the display can be moved by clicking and dragging with the mouse Drill mode can also be used where the region highlighted by a mouse click and drag will be drilled into once the mouse button is released Jumping Around You can jump to a position in t
48. is Dashboard select the Sample and then choose Browse Sample s This will open the Genome Browser centered on the target you have defined in the Analysis Experiment You can start to analyse the variants From the Sample Dashboard select Analyse sample variants which will start a new dashboard style screen with the same name In here you can the variants that have been called in lists that are split by on target and off target lists You can select items in these lists and then Jump to Variant which will move the Genome Browser and show you the variant and short read reads at that position From here you can also start the Approval Dashboard in order to approve or reject variants for further analysis or you can start the Report Dashboard to view summary reports for this Sample Custom Profile If none of the built in Profiles are useful then you will need to create your own custom Profile within the tool The first thing to note is that the Profiles are essentially just convenience wrappers around some Reference sequences and a Target definition So to create the equivalent of a new Profile all you need to do is to import some reference data and possibly some known variants for that data and then create a new Target based on that reference data Even having a Target is not mandatory but using one is recommended as there are lots of Target based functions within the tool This tutorial will walk you through the steps needed to do
49. is created data can be imported for analysis The other way to create Samples is to use the Import multiple samples function from the Analysis Dashboard which will both import fastq and automatically create Samples at the same time Map and Align Samples If you have imported sequencing data then you can map and align this data to a Reference Genome using this function This version of the Map and Align runs with multiple samples i e all the samples selected in the list visible in the Analysis Dashboard If any of the samples selected already have mapped data and or variant calls they will be silently ignored This function has three analyses built in to it Map and Align using Omixon mappers e Variant Call using GATK optional e Quality Control coming soon The Map and Align step will always be performed The Variant Call and Quality Control steps are optional The Variant Call can be performed later using the Call Variants function or the mapped data can be copied elsewhere and other variant caller used instead The Quality Control cannot be run separately only as part of the Map and Align process The steps in this wizard Select Reference Data Select Species Select Sequencer Advanced Options Variant Call Options Quality Control Options coming soon Like all the wizards within the tool most of the options within this wizard will already be pre filled and pre selected based on your chosen Analysis and
50. mixon Target User Manual Finally the installer informs you about the outcome of uninstall Omixon Target Application 1 0 0 Uninstall Omixon Target Application Uninstall Omixon Target Application was successfully removed from your computer Finish Pro Data Analysis Module installation notes Introduction The Pro Data Analysis Module is an optional Module which supports additional features compared to the Standard Data Analysis Module The Pro Module is supported only on a 64 bit Linux platform The Pro Module uses several well known bioinformatics tools behind the scenes You need to install and configure these tools manually once they are on the PATH they can be used by Omixon Target General notes and configuration If you have all the tools installed and available on your PATH then no additional configuration is needed If you don t have some tools on your PATH you have to configure them manually for Omixon Target to let the application find them The configuration file is called tools properties and located in the proserver etc folder of your installation directory There you can find a configuration line for each tool and you can provide the full path to the executable of the tools List of tools Here is the list of tools to be installed in your environment to get all Pro features working Samtools Homepage of the tool http samtools sourceforge net Version used for testing 0 1 8 Installation with package ma
51. mixon Target i e it has none of the data analysis features The HLA edition has no up front license fee The HLA Typing Module offers two licensing schemes 1 A credit based pricing scheme with no up front license fee with limited use one credit is consumed per sample analysed more credits can be purchased on demand 40 free HLA typing credits are included in the evaluation version 2 An annual fee based license which allow unlimited HLA Typing Please contact sales omixon com for a quote The HLA Module works on Windows 64 bit recommended Linux 64 bit recommended and Mac OS X Adding and removing Modules Note that it is possible to add and remove Modules to and from your Omixon Target installation The Modules are controlled by a license file so if you would like to alter your installation then you should contact Omixon and we can generate a new license file for you which can be imported within the Settings function in the tool Key Concepts Target The data analysis part of the tool is built around the concept of an analysis target This can currently be one or more genes exons or amplicons There is a Target Dashboard to manage the targets within the tool The annotations for a Target can be imported directly from a bed or gff file While using a target is recommended it is not actually mandatory within the tool Hierarchy of Experiment Analysis and Sample In order to help with organising the data belonging to the s
52. mple or family trio comparative studies Discovery Omixon also use the tool to support collaborative genomic biomarker discovery projects Sequencing Technologies Omixon Target supports sequencing data from these major sequencing technologies e Illumina e lon Torrent e Roche 454 Variant Calling The only Variant Caller that is available in the Standard edition is a GATK pipeline using the Broad Institute GATK variant caller This gives excellent results with Illumina data There is a better variant calling pipeline for lon Torrent or Roche 454 data available within the Pro edition HLA Typing So far most of the validation that has been done on the prototype HLA Typing algorithms has been done with paired Illumina data While it is possible to get results with other kinds of data this is not yet reached an equivalent standard as using the paired Illumina data The HLA Typing results for the other platforms will be improved in future releases Modules Omixon Target has a modular structure Omixon Target consists of a main Genome Browser Module with some optional Modules This means that Omixon Target can simply be used as a Genome Browser if desired i e for visualization of NGS data analysis results in SAM BAM and VCF formats Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Genome Browser Module This is always included as standard with Omixon Target It includes some data management function
53. n Gene Amplicon Press Next e Select a bed or gff file from the file system containing the annotations that describe your Target Press Finish Import Multiple Samples This function is available from the Analysis Dashboard If you have short read data from sequencing run you can import it with this function and then run the Map and Align function which will align the data to a Reference Genome and run a variant call in order to identify the variants It is essentially identical to the Import Sequencing Data function within the Sample Dashboard the goal it to import single or multiplexed fastq sff data into the tool The difference with this version is that it will do two things e Import fastq sff data or paired fastq data e Automatically create a new Sample for each imported fastq file or pair of fastq files in case of single sample data e Demultiplex each fasta sff file and create a new Sample for each sample found in the files identified by barcodes This function will allow you to import multiple fastq sff files or multiple pairs of matching fastq files at the same time The second file is optional and is for paired reads i e if your analysis produced paired end or mate pair data If using paired reads the two input files are assumed to have the exact matching reads in the exact same order there is currently no in built check for this The paired read import is not supported for sff files Note that the sff import
54. n source bioinformatics tools These are not however bundled within the Omixon Target application The IMGT HLA database is used for the HLA typing here are the citations e Robinson J Mistry K McWilliam H Lopez R Parham P Marsh SGE The IMGT HLA Database Nucleic Acids Research 2011 39 Suppl 1 D1171 6 e Robinson J Malik A Parham P Bodmer JG Marsh SGE IMGT HLA a sequence database for the human major histocompatibility complex Tissue Antigens 2000 55 280 287 Tutorials First Use The easiest way to get started with Omixon Target is to choose a pre configured Profile to work with or more than one Each Profile already includes a reference and a target Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Fast Start e Add Profile Import sequencing fastq data e Map the reads and call variants e Analyse the results This tutorial only explains the steps required for the Fast Start There are other tutorials available for using the Expert profile and setting the tool up manually Adding a Profile The first step of the Fast Start involves adding a Profile the alternative is to create your own custom Profile using Create Profile see the next tutorial for details This function is available from the Data Analysis Dashboard Adding a new Profile will start a new background task which by default will download and install all the data required to use the Profile It will also create a new Expe
55. nager on Debian based systems apt get install samtools Bowtie Homepage of the tool http bowtie bio sourceforge net index shtml Version used for testing 0 12 8 Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Installation with package manager on Debian based systems apt get install bowtie FASTX Toolkit Homepage of the tool http hannonlab cshl edu fastx_toolkit Version used for testing 0 0 13 2 Installation with package manager on Debian based systems apt get install fastx toolkit Seqtk Homepage of the tool https github com Ih3 seqtk Revision used for testing 771d60b8f774482a77d60dd8559d 1 7bd487c56e8 Installation from source git clone https github com h3 seqtk git cd seqtk make cp f seqtk usr local bin Biopython Homepage of the library http biopython org wiki Biopython Version used for testing 1 54 Installation with package manager on Debian based systems apt get install python biopython Acknowledgements Collaborators We would particularly like to thank SmartArt who prepared all the graphics and images used within the tool Third Party tools The Genome Analysis Toolkit GATK from the Broad Institute is used for the variant calling an older version 1 6x is used A number of tools from SamTools Picard are also used within the tool for handling and manipulating SAM and BAM files The Pro Data Analysis Module allows access to a number of commonly available ope
56. ng Kft Omixon Target User Manual Application Setup Wizard Setup has finished installing Omixon Target Application on your computer The application may be launched by selecting the installed icons Click Finish to exit Setup After installation a startup icon named OmixonTargetApplication can be found in the Applications folder G Applications m m FAVORITES ooth Preview QuickTime Playe 5 Dropbox E All My Files o AirDrop PAN Applications E Desktop ferences OmixonTargetAppl Time Machine ication Documents da N ZIN In Mac OS X the uninstallation process is straightforward the user has to just send to Trash by the application icon Linux installation guide We provide two versions of installer package for Linux operating systems e 64 bit with Java Runtime environment e 32 bit with Java Runtime environment The installer package which contains the Java Runtime environment contains a 64 bit JRE and it is recommended for 64 bit operating systems In case Omixon Target really needs to be run in a 32 bit environment it can be done by choosing the appropriate installer Note that 32 bit operating systems are not officially supported not fully tested but that does not mean that the application cannot run The current limitation of 32 bit operating systems is mostly related to the amount of memory available there The install packages are single file shell scripts This typ
57. nish This will start a new background task Once this task has completed you can create a new Experiment and start using your new Target New Target The first thing to mention is that you don t have to use a Target However a number of the features within the tool are built to support the Target concept and having a well defined target can lead to better analysis results There are two ways to create a new Target within the tool The first and easiest is to import a set of Target annotations using a gff or bed file The second way is to manually import annotations for a reference and then manually create a Target to use those annotations Import Target You should start at the Data Analysis Dashboard e Click on Targets and choose the Import Target button from the menu above the list of Targets e Give your new Target a name e Select the Annotation Type Amplicon Exon or Gene e Select the bed or gff file from the file system e Choose the Reference e Select the Contigs e Press Finish This will start a new background task Manual Target Creation There are three steps involved with creating a new Target the first of these is actually performed with the Reference Genome that will be used For the purposes of this tutorial it is assumed that the Reference Genome has already been created and configured correctly see the New Reference tutorial for more on this Reference Genome configuration via the Reference Genome Dashboar
58. not to run a GATK variant call after the map and align function has run You can always run the variant call later if desired The dbSNP known variants file is an optional parameter You can either use dbSNP data that has been imported into the tool or select a dbSNP file from the file system In the OmixonTarget Pro edition you can also choose to run a Samtools mpileup variant call instead of a GATK variant call This is recommended for 454 and lonTorrent data Call Variants If the Map and Align function is used to map short reads then there is an option to run a Variant Call within that wizard If that option is not chosen or if already mapped reads are imported into the tool then a separate Variant Call can be started The Variant Call invokes a full GATK pipeline following the recommended best practises of the Broad Institute Like all wizards in the tool as many options as possible are already preselected within the wizard These are the wizard steps e Select Genomic Data Select Species Select Sequencer Advanced Options Variant Call Options e e e Advanced Options e Parameters file This is currently not used for the variant caller Variant Call Options The dbSNP known variants file is an optional parameter You can either use dbSNP data that has been imported into the tool or select a dbSNP file from the file system In Omixon Target Pro edition you can also choose to run a Samtools mpileup based varian
59. o the assignments separately for each sample allele you can find similar functions on the HLA Typing sample and allele result screens The results can be exported in TXT tab delimited text CSV comma separated text or XLS Excel format Tip If you would like to export only the assigned allele candidates click the Assigned Only button then export the results You can filter the displayed loci by using the Setup Loci function HLA Typing Allele Result A more detailed view of the HLA Typing results for a single Allele For each allele candidate we supply e Detection A percentage value based on a comparison of the density of coverage The allele uses the lowest detection value among its exons High values are better e Average coverage The average number of short reads covering the whole exon or allele exons with zero coverage are not counted e Exons covered Number of exons with non zero coverage number of available exon sequences for the allele candidate e Detection and Average coverage values for each covered exons A minimum coverage threshold can be set for the allele result table This limit can be set as an absolute coverage value i e the mean number of reads for the allele or a relative coverage threshold minimum coverage relative to the top allele The default value for minimum coverage is 10 for the absolute and 95 for the relative coverage filter function Alleles below the coverage limit are shown as
60. ot of the currently visibly tracks within the Genome Browser This will create a png file at your chosen location Analysis Results Summary statistics for all the Samples in the Analysis This is a very simple summary screen that is available from the Analysis Results button in the Analysis Dashboard It displays overview statistics of the progress for all the Samples in the Analysis References Centralised reference configuration for the whole tool Omixon Target has a centralised configuration for the References reference sequences used in the Data Analysis portion of the tool These References can be set up once and then used in multiple Experiments and Analyses This screen lists the available Reference Genomes and allows Reference Genomes to be created or deleted Clicking on a Reference Genome will take you to the Reference Genome Dashboard where reference data in fasta format can be imported along with known variants from dbsnp Targets Centralised Target configuration for the whole tool Omixon Target has a centralised configuration for the Targets essentially lists of annotations used in the Data Analysis portion of the tool These Targets can be set up once and then used in multiple Experiments and Analyses Targets are actually optional but recommended This screen lists all the Targets defined and allows Targets to be created or deleted It is possible to create a Target by importing annotations in BED or
61. ou would like to download Option 2 Using the Create Custom Profile function If you would like to create a new Profile not based on the HG19 reference genome then this wizard is the easiest way to achieve that This wizard is very similar to the Create HG19 Profile wizard above The main difference is that you can either select an existing reference genome or create a new one during the wizard steps You can manually create a new reference genome see steps below before starting this wizard if you wish This will start a new background task Once this task has completed you should see your new Experiment and Analysis and you can start importing sequencing data and using your new Target Option 3 Manually Creating a Profile You can also manually recreate the Create Custom Profile steps using the individual functions Create a Reference You should start at the Data Analysis Dashboard e Click on References and then click on Create New Reference e Select the name for your reference and species e Press Finish Importing Reference Data Omixon Target needs individual chromosomes to be added the whole Human Genome multifasta file will not work correctly within the tool You should start at the Data Analysis Dashboard e Click on References and then click on the new custom reference you just created in the list e Select the Import reference data item in the Actions menu on the left e Choose the reference chromosomes
62. plicons The annotations belong to a particular Reference Genome and can be imported into the tool via the Reference Genome Dashboard List of wizard steps e Create Target It is not sufficient to simply create a Target once a Target has been created it should be configured by clicking on it which will navigate to the Target Dashboard and then configuring the Target via the Configure Target wizard Configure Target This function can be used to add and remove annotations from a Target The annotations will already need to exist either imported against a Reference from the References screen or by using the Import Target Annotations function in the Targets screen or the Create Profile function in the main Data Analysis screen Import Target Annotations This function will import annotations for genes exons or amplicons and use them to create a Target The reference genome will need to be created first if it doesn t exist already and all the reference data should already be imported within Omixon Target The easier alternative to this function is to use the Create Profile wizard which merges the Import Reference Data Import Known Variants and this Import Target wizard into one function Export Mapped Data Any mapped short read data that has been mapped within Omixon Target or imported using the Import Mapped Data function can be Copyright 2012 Omixon Biocomputing Kft Omixon Target User
63. port mode is not supported A background task will be started and once it s finished a new item will appear in the main list within the HLA Typing Dashboard where you will be able to see the results of the HLA Typing Note that the typing process has different categories and which could be executed depending on your license credits The license category can be selected on the first page of the wizard Paired Read Options e Minimum Distance the minimum expected distance between the two reads e Maximum Distance the maximum expected distance between the two reads e Orientation the orientation of the reads with respect to each other The first file is the first in the orientation as well Options are FR forward reverse i e first file contains forward reads second file contains reverse reads RF reverse forward and FF forward forward Advanced Options These options dicatate where the data has come from and effect how the underlying algorithms deal with the data Some data sources e g whole genome and whole exome are more noisy and the algorithms can help to filter out this extra noise e Choose what the source is of the HLA Typing data Maximum Reads Processed This is very important This dictates how many reads or how many pairs will be processed from each input file in order to do the HLA Typing You can now choose from a pre configured set of typical sequencing runs including Whole Genome and Whole Exome You can also choos
64. r a sample or an allele or a short read alignment visualisation can be viewed for that Sample by pressing the Sample Details the Allele Details or the Browse Sample buttons respectively A minimum coverage threshold can be set for the summary result table This limit can be set as an absolute coverage value i e the mean number of reads for the allele or a relative coverage threshold minimum coverage relative to the top allele The default value for minimum coverage is 10 for the absolute and 95 for the relative coverage filter function Alleles below the coverage limit are shown as empty cells Note that allele candidates with a lower coverage than the selected limit are NOT shown on the HLA Typing sample result dashboard which contains the detailed results For each allele one or more allele candidates can be assigned There are four different ways to assign allele candidates 1 Candidates can be assigned manually by clicking on the checkmark before the allele candidate s name 2 All winner candidates i e the best allele candidates based on coverage statistics can be assigned using the Assign Winners button 3 All unambiguous results can be assigned using the Assign Unambiguous button 4 All allele candidates can be assigned using the Assign All button All assignments can be deleted by the Unassign All button Note that the Assign buttons on this screen affect all the displayed samples if you prefer to do the assignmen
65. riment and Analysis for you which will be pre configured with the sequencer chosen and with the reference and target from within the Profile After this Add Profile task has finished it will take 5 to 10 minutes depending on the speed of your internet connection you can move to step 2 of this Fast Start tutorial Download problems If for some reason you cannot download files from within the tool e g due to network security settings you can download the reference files manually and use the Select Local file s option instead of the Download file s option in the wizard The files for the in built profiles can be downloaded from here BRCA profile e http omixon download s3 amazonaws com target_ref_hg19_chr13 zip e http omixon download s3 amazonaws com target_ref_hg19_chr17 zip HLA profile e http omixon download s3 amazonaws com target_ref_hg19_chr6 zip CFTR profile e http omixon download s3 amazonaws com target_ref_hg19_chr7 zip Data Analysis Example Data Data Analysis is an optional Module built into Omixon Target We offer a few example datasets that go with the in built profiles These allow you to easily try out the Map and Align and Call Variants functions within the tool BRCA profile example data paired illumina data two fastq files http omixon download s3 amazonaws com target_brca_example zip HLA profile example data ion torrent data single fastq file and paired illumina data t
66. rity settings you can download the necessary files manually and use the Select Local file s option instead of the Download file s option in the wizard For each profile the setup files are packaged by chromosome and can be downloaded from the following links BRCA profile e http omixon download s3 amazonaws com target_ref_hg19_chr13 zip Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual e http omixon download s3 amazonaws com target_ref_hg19_chr17 zip HLA profile e http omixon download s3 amazonaws com target_ref_hg19_chr6 zip CFTR profile e http omixon download s3 amazonaws com target_ref_hg19_chr7 zip Create HG19 Profile The tool starts up in Expert mode with only the Expert Profile configured and no reference data imported or targets set up A Profile is simply a wrapper around a Target plus a set of reference data and known variants A Target is simply a list of reference annotations for either genes exons or amplicons It is possible to add a pre configured Profile to the tool using the Add Profile function or you can use this Create HG19 Profile option instead It s also possible to skip Profile set up entirely and simply configure the tool manually e Fill in the details Profile Name Target Name Experiment Name Analysis Name A new Profile Target Experiment and Analysis will be created Press Next Choose one or more sequencers for your Analysis tip use C
67. s as well as a full featured NGS Genome Browser and some tables for variant visualiziation The Genome Browser Module works on Windows Linux and Mac OS X Standard Data Analysis Module Optional This module includes a range of general genomic data analysis algorithms including Omixon s own aligner and a full GATK variant call pipeline It also features an approval work flow for automated or manual filtering and approval of variants The Standard Module works on Windows 64 bit recommended Linux 64 bit recommended and Mac OS X Pro Data Analysis Module Optional This includes all the features of the Standard Data Analysis Module including all the standard genomics data analysis algorithms and the approval work flow This Module is an alternative to the Standard Data Analysis Module for Linux users In addition it also includes integrated access to a number of third party tools including a better variant call pipeline for lon Torrent and Roche 454 data based on the SamTools mpileup and some supporting scripts a demultiplexing import and an sff import feature The BWA aligner for Illumina data and a simulation based Quality Control feature will be coming soon The Pro Module is only available for a 64 bit Linux operating system and requires some manual installation and configuration of third party tools in addition to the usual installation steps HLA Typing Module Optional This Module contains only the HLA Typing feature of O
68. t call instead of a GATK pipeline This is recommended for 454 and lonTorrent data Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual Auto Approve Even with targeted sequencing there can be a large number of variants to deal with Auto approve allows the bulk approval of variants that meet one or more of a number of criteria including e The quality of the call is above a minimum quality e The coverage at the location is greater than a minimum coverage e The call is on Target Auto Reject Even with targeted sequencing there can be a large number of variants to deal with Auto reject allows the bulk rejection of variants that fail to meet one or more of a number of criteria including e The quality of the call is above a minimum quality e The coverage at the location is greater than a minimum coverage e The call is on Target Auto Reset If you have made a mistake with one of the Auto Accept or Auto Reject functions you can reset all your variants back to pending status by using the Auto Reset function This is an all or nothing operation Design Analysis This wizard usually only has to be run once when a new Analysis is created This wizard inherits all its settings from the parent Experiment and can be used to restrict the options from the Experiment for each Analysis The steps in the wizard General Properties Choose Sequencer Choose Target Choose Species Choose Genome Choose Contig
69. tes can be hidden Analysing result Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual By clicking on the Analyse Result button the HLA Typing allele result screen is opened This page contains detailed exon level statistics for the allele candidates Note that only samples visible in the current browser session will be displayed in the allele result table Track Setup You can configure which tracks you would like to see in the display Display Setup You can display hide read pair information indels SNPs and soft clips Exporting HLA Genome Browser Data You can Copy to Clipboard the details of individual selected items in the display You can also Capture a Screenshot of the currently visibly tracks within the HLA Genome Browser This will create a png file at your chosen location User Management User Management allows Users to be added edited and removed from the system There must be at least one Super User at all times The first registered user automatically becomes the Super User this user cannot be deleted Configure Application This wizard allows you to set up a mail server via SMTP for sending bug and error reports directly to Omixon You can adjust the colour scheme of the Genome Browser here The zooming resolution of the Genome Browser can also be set on this screen by changing the Overview factor This is a the maximum number of nucleotides displayed per pixel The
70. time environment e a 64 bit version named omixon target_windows x64_1_0_0 with_jre exe 232 bit version named omixon target_windows_x86_1_0_0 with_jre exe The only fully tested operating systems are the 64 bit ones The installer package which contains Java Runtime environment contains a 64 bit JRE and it is recommended for 64 bit operating systems omixon target_windows x64_1_0_0 exe After running the installer the following dialog should appear Setup Omixon Target Application 1 0 0 Welcome to the Omixon Target Application Setup Wizard This will install Omixon Target Application on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup The next step is the destination directory selection Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual a Setup Omixon Target Application 1 0 0 E x Select Destination Directory Where should Omixon Target Application be installed Select the folder where you would like Omixon Target Application to be installed then click Next Destination directory C Program Files omixon target Required disk space 124 7 MB Free disk space 202 976 MB install4j By default the Program Files directory is selected in addition to that an application directory is appended After selection by clicking on the Next button the installation process is followed
71. ts separately for each sample allele you can find similar functions on the HLA Typing sample and allele result screens The results can be exported in TXT tab delimited text CSV comma separated text or XLS Excel format Tip If you would like to export only the assigned allele candidates click the Assigned Only button then export the results At this level the summary HLA Typing results can be displayed in 4 6 or 8 digit formats In the more detailed results always the 6 or 8 digit format can be seen Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual You can filter the displayed loci by using the Setup Loci function HLA Typing Sample Result A more detailed view of the HLA Typing results for a single Sample For each allele at each locus we supply e Detection A percentage value based on a comparison of the density of coverage The allele uses the lowest detection value among its exons High values are better e Average coverage The average number of short reads covering the whole exon or allele exons with zero coverage are not counted e Exons covered Number of exons with non zero coverage number of available exon sequences for the allele candidate By clicking on the Browse button next to the name of a specific allele the HLA genome browser can be opened This HLA visualisation tool shows the short reads in the Sample aligned to the allele candidates The best allele candidate is m
72. u can start to use the HLA Typing function Download problems If for some reason you cannot download the HLA configuration file from within the tool e g due to network security settings you can download the files manually and use the Select Local file s option instead of the Download file s option in the wizard The in built HLA configuration file can be downloaded from here Copyright 2012 Omixon Biocomputing Kft Omixon Target User Manual http omixon download s3 amazonaws com target_haplotype_db zip HLA Typing This function is available from the HLA Typing Dashboard Before running HLA Typing the Setup Typing wizard will need to be run You need to fill in a name for this HLA typing Analysis plus select fastq files for the input you can select multiple files and multiple paired files You also need to select species and which sequencer was used to created the fastq data This function will allow you to process multiple fastq files or multiple pairs of matching fastq files at the same time The second file is optional and is for paired reads i e if your analysis produced paired end or mate pair data If using paired reads the two input files are assumed to have the exact matching reads in the exact same order there is currently no in built check for this You can also use SFF files as input or you might combine FASTQ and SFF files as well Note that if any of the input files is in format SFF then paired im
73. ualised for each allele candidate include e Reference Sequence e Exon Annotations e Short Reads e Coverage By default short read alignments are displayed in a stranded fashion so that forward strand reads pink and reverse strand reads yellow can be easily distinguished within the display The Display Strand function turns this on and off The short reads track can be collapsed which gives a summary view of the short reads and does not allow each read to be inspected in detail Individual items exons short reads within the HLA Genome Browser can be selected and it s possible to browse elsewhere within the same locus and then jump back to the selected item or copy the details of the selected item to the clipboard Expanding the display At the top left of the tool is a small control that allows an expanded view This actually works for all screens in the tool but is most useful for the HLA Genome Browser Clicking the same icon again will go back to the usual display Rotating the Genome Browser The vertical HLA Genome Browser view is more useful for comparing multiple allele candidates The view can be rotated to use a horizontal display which is a bit more traditional for Genome Browsers and is more useful when browsing a single allele candidate Zooming By default the Genome Browser starts in Drag mode where the mouse wheel or and keys can be used to zoom in and out and the display can be moved by clickin
74. wo fastq files http omixon download s3 amazonaws com target_hla_example zip In order to import the fastq you should use the import multiple samples function from the Analysis Dashboard select Go on the appropriate Analysis after Add Profile For the paired data you need to select the first file 1 fastq in the first file selector in the wizard and the second file 2 fastq in the paired data file selector The default pair parameters orientation distance are fine for the example data sets Once you have imported the fastq you will be given the option to run the Map and Align function for the Sample Import sequencing fastq data Once the Add Profile task from step 1 has finished you can start to work with the Profile The first step is to select a Sample to work with There is a small data management hierarchy within the tool the Samples are grouped together with an Analysis and the Analyses are grouped together within an Experiment Assuming that you selected to also Import Example Data while adding the profile there will already be an example Experiment for you to work with Select the example Experiment by clicking on it which will navigate to the Experiment Dashboard and select the example Analysis in the same way moves to the Analysis Dashboard You can click on the Create Sample button in the Analysis Dashboard and create a brand new Sample to work with Copyright 2012 Omixon Biocomputing
75. ysed within the tool Once variants have been called there are a number of downstream analysis steps that can be performed including browsing the results in the Genome Browser analysing the results within the Analyse Genome Dashboard approval of variants via the Approval Dashboard and creating and viewing reports in the Report Dashboard Mapping Alignment Variant Call These functions are only available within one of the optional Data Analysis Modules Standard or Pro One of the primary goals of the tool is to identify variants within the sample The tool includes sequencer specific algorithms for mapping and aligning NGS short reads against a reference sequence and then calling variants against the aligned short reads using a GATK pipeline following the recommended best practises of the Broad Institute The mapping and alignment algorithms used are currently Omixon s own There are separate algorithms for Illumina 454 and lon Torrent data which include different error models for the different sequencers One future plan is to run not just Omixon s algorithms but some other alignment algorithms as well and to compare and or merge the results of this double alignment It is also possible to simply import already mapped short read data and just run a variant call or even just import already called variants into the tool for downstream analysis Genome Browser There is a genome browser included in the tool This allows you to brows
76. ysis Omixon Target has a centralised configuration for the References reference sequences used in the Data Analysis portion of the tool These References can be set up once and then used in multiple Experiments and Analyses This screen lists the Reference Genomes linked to this Analysis and allows Reference Genomes to be created or deleted Clicking on a Reference Genome will take you to the Reference Genome Dashboard where reference data in fasta format can be imported along with known variants from dbsnp Analysis Targets Configure Targets for an Analysis Omixon Target has a centralised configuration for the Targets essentially lists of annotations used in the Data Analysis portion of the tool These Targets can be set up once and then used in multiple Experiments and Analyses This screen lists the Targets attached to this Analysis and allows Targets to be created or deleted It is possible to create a Target by importing annotations in BED or GFF format using the Import Target function Clicking on a Target will take you to the Target Dashboard where the Target can be configured HLA Typing Analysis Result A high level overview of the HLA Haplotying results A high level overview of the HLA Typing results for a single Analysis i e for a single submission of the HLA Typing tool or for multiple Analyses This can include the summary results for many samples Each Sample can be selected and the detailed results fo

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