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Omixon Target User Manual

1. 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 GFF format using the Import Target function Clicking on a Target will take you to the Target Dashboard where the Target can be configured Create Target Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 amplicons 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
2. DO NOT AGREE TO ALL OF THE TERMS OF THIS AGREEMENT CLICK THE DO NOT install4j Select the path where you would like the application to be installed Select Destination Directory Where should Omixon Target be installed Select the folder where you would like Omixon Target to be installed then click Next Destination directory C Users OmixonTargetUser OmixonTarge Required disk space 140 0 MB Free disk space 77 827 MB Select data directory where the permanent data files will be stored Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Data directory The directory where the application will store its permanent data files This directory should be readable and writable by all users of the application It should have enough space to store large amount of genomic mapping and other data C Users OmixonTargetUser oqve Select temporary files directory Temp directory The directory where the application will store its temporary data files This directory should be readable and writable by all users of the application It should have enough space to store large amount of genomic mapping and other data C Users OmixonTargetUser AppData Local Temp Enter name for Start Menu item Use the checkboxes to make it visible for all users of the operating system or to not create Start Menu item at all Copyright 2013 Omixon Biocomputing Kft Omix
3. If you are running a 32 bit version and or you don t have more than 5GB of memory available the other option is to reduce the sensitivity of the mapping tool You need to create a text file and to add the following line orm sampling default 4 Save it then load it as a Parameters file by using the Advanced Parameters tab of the Map and Align wizards 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 try a smaller value such as orm sampling default 2 This will save about 30 40 of the memory required Overview Home Dashboard Introduction This is main home page for Omixon Target There are only four functions available from this screen e Data analysis e HLA Typing e Sandbox Genome Browser e Settings The Data Analysis module is for general analysis of sequencing targets where sequencing data can be mapped and aligned against reference sequences and variants can be called visualised and analysed It s an optional module with a separate license The HLA Typing module is only for determining and visualising the HLA types within a set of NGS sequencing data It is simple fast and accurate It s an optional module with a separate license Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual The Sandbox Genome Browser module is for quick visualisation of alignments va
4. 15400 wizard can be opened by pressing the CTRL and J keys on the keyboard After the wizard is opened you can type in a single coordinate or a pair of coordinates and you can specify a reference contig When a reference contig is not chosen the currently shown contig is used Coordinates can be specified with or without thousand delimiters i e 15200 15 200 and 15 200 are all accepted and mean the same thing e CTRL J chr2 15 200 ENTER gt The Genome Browser display will jump to chr2 15200 Jump to an annotation The Jump To Annotation wizard can be opened by pressing the CTRL and F keys on the keyboard After the wizard is opened you can type in the name or the beginning of the name of an annotation e g gene exon amplicon When the specified search term is ambiguous i e more than one annotation name starts with the specified string a list of annotations is presented and the desired annotation can be selected from the list The input field in this wizard is not case sensitive i e brcal and BRCA1 are both accepted and mean the same thing Search for a nucleotide sequence By pressing CTRL S you can open the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible reference contig and then step through the hit positions Shortcuts Keyboard mouse navigation options and shortcuts UP DOWN arrow keys scroll alongside the reference contig LEFT RIGHT arrow keys s
5. 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 e and the sequencer used for the sequencing of the data can be done at both Experiment and Analysis level 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 cause the data to be downloaded and automatically configured with the tool It will also create and configure a starting Experiment Analysis and Sample Reference Genomes One or more reference
6. Chromosome 17 e http omixon download s3 amazonaws com target_ref_hg19_chr17 zip etc 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 Exon Gene Amplicon Press Next e Select a bed or gff file from the file system containing the annotations that describe your Target e Sele
7. Server Manager The file is provided by the system administrator who needs to export the connection settings into a file by clicking Export on the selected connection in Server Manager Acknowledgements Collaborators We would particularly like to thank SmartArt who prepared all the graphics and images used within the tool Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 open 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 Burrows wheeler aligner software package for mapping low divergent sequences against a large reference genome e The short read alignment component bwa short has been published Li H and Durbin R 2009 Fast and accurate sho
8. 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 Best Match Warning Flags e Alleles displayed with the blue font are homozygous e Alleles displayed in italics are imbalanced i e present in a much smaller proportion of the reads that the main candidates Results are annotated with warning flags Some of these flags apply to single allele candidates others apply to the pair of candidates at a particular locus Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Single candidate flags e Allele candidates with lower than 90 detection on exons 2 and 3 Class genes or exon 2 Class II genes get an error flag red The detection is the proportion of the exon with sufficient coverage This flag indicates that there may be some key areas of the candidate not well covered by short read data so the result should be treated with caution and may require manual inspection e Allele candidates with lower than 98 detection on exons 2 and 3 Class genes or exon 2 Class II genes get a warning flag orange e If rare alleles are not ig
9. with the left mouse button The selected annotation element is marked by a lighter colour The attributes of the annotation element can be edited using the Edit selected annotation function this button is located on the middle bottom panel next to the Create new annotation button You can also open the annotation editor screen by pressing the ENTER button after selecting an annotation e To resize an annotation entry in the browser select the annotation lock the cursor you can do this by pressing Space on Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual your keyboard then press the Resize selected annotation button on the middle bottom panel or press R on the keyboard The selected annotation will be resized to end at the cursor position e To move an annotation entry in the browser select the annotation lock the cursor you can do this by pressing Space on your keyboard the press the Move selected annotation button on the middle bottom panel or press M on the keyboard The selected annotation will be moved to the cursor position Deleting tracks To delete an annotation entry select the annotation then press the Delete selected annotation button red X symbol on the middle bottom panel or the Delete button on your keyboard Showing or hiding tracks All tracks both built in and user created tracks can be temporarily hidden using the Hide track s button in the Track Ma
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11. 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 Options are FR forward reverse RF reverse forward and FF forward forward Advanced Options These options dictate 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 Process all reads 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 e Read processing option You can now choose from a pre configured set of typical sequencing runs including Whole Genome and Whole Exome You can also choose Custom to manually set how many reads to process If you are not sure then use the All option to process all the reads e Maximum Reads Processed This is very important You may need to experiment with this value to find the best match for your data This dictates how many reads or how many pairs will be processed from each input file in order to do the HLA Typing For very targeted data sets including only the HLA loci this value doesn t usually need to be too high about 20 000 For larger kits such as the RainDance HLAseq kit the whole HLA super
12. T Ubuntu One Music Universal Access Wine Uninstalling the product If you want to remove Omixon Target an uninstaller script is available in the directory where the application was installed If you intend to use future version of Omixon Target and you don t want your work data to be deleted keep the checkbox unchecked If you wish to cleanup the working directory then selet the option Yes Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Omixon Target 1 4 0 Uninstall Select Additional Tasks al Which additional tasks should be performed r Select the additional tasks you would like Setup to perform while uninstalling Omixon Target then click Next Yes Do you want to remove your working data Next gt Cancel Click Next to proceed with the uninstallation process Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Are you sure you want to completely remove Omixon Target and all of its components Click Next to continue or Cancel to exit Setup Next gt Cancel Finally the installer informs you about the outcome of uninstall Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Omixon Target was successfully removed From your computer Finish This symlink is useful for users who usually start the application from the command line Since the installer is cr
13. This function will import doSNP known variant annotations for the Reference Genome Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 Variant Effect In this wizard an SnpEff variant effect prediction database can be added for a reference The database must be in the format specified by the SnpEff documentation If a variant effect database file is added the entries required for the SnpEff run are automatically created in the snpEff config file In any built in profiles are added or HG19 based profiles are created the HG19 SnpEff database is automatically downloaded and added to the reference Tip Custom databases can be created for any kind of reference sequence For details take a look at the SnpEff documentation Import Annotations This function will import Gene or Exon annotations for the Reference Genome This will be used later for Target configuration Currently bed and gff formats are supported If you need another format please let us know The steps in this wizard are e Select Annotation Type e Select Input Files Select Contig Import Target Annotations This function will import annotations for genes exons or amplicons and use them to create a T
14. allele Browse Sample a short read alignment visualisation 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 first 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 the 6 or 8 digit format can be seen Interpreting Results By default only the best matching candidates are displayed in the results table These are the pairs of candidates with best overall combination of detection and average coverage scores If an ambiguous best match is returned then there will be more than two best matching candidates for the locus By pushing the Best Matches Only button again it is possible to see other close allele candidates The Setup Filters option can be used to display more or less allele candidates By selecting a sample clicking on the table you will be presented with more options for drilling down into the details of the results You can see details for a sample for a particular allele you can browse allele candidates in the Genome Browser and you can see the pair of candidates for each locus More help is available within each of these functions Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Detection The proportion of the allele that
15. 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 Display setup The visualisation of variants in the short read track can be customized as well Variants are always visible on the nucleotide level but insert and deletion marks can be selectively turned on and off Soft clips can be shown or hidden as well Reads can be shown in normal mode or paired mode in paired mode reads are sorted differently and pairs are connected with a thin line Exporting Meta data and Creating Screenshots You can Copy to Clipboard the details of individual selected items in the display You can select a read and by clicking on the Copy to clipboard button which can be found on the middle panel in the bottom of your screen you can copy the name and CIGAR string of the selected read to the clipboard Similarly for a selected annotation the position and name of the annotation can be copied to the clipboard You can also copy the reference sequence belonging to a selected item read or annotation by clicking on the Copy reference to clipboard button You can also Capture a Screenshot of the currently visibly tracks within the Genome Browser This will create a png file at your chosen location Working with Tracks Tracks used in the Sandbox Genome Browser There are two different kinds of tracks used within the Sandbox Genome Brow
16. be automatically downloaded during installation Installation steps Open the installer which is packed within a DMG archive Click on the Omixon Target Installer app icon 000 Downloads ER Wa gt PB om 27 2 Lm le a Back View Action Arrange Share Path New Folder Delete Get Info Search FAVORITES Name Date Modified Size Kind E All My Files omixon target_macos_1_4_0 dmg Today 4 56 PM 36MB Disk Image gt AirDrop ooo L omixon target A Applications E Desktop A Documents Omixon Target pc a Movies J Music Pictures Ai omixontargetuser After launching the installer Welcome dialog of the setup wizard appears Setup Omixon Target 1 4 0 Welcome to the Omixon Target Setup Wizard This will install Omixon Target on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup Cancel Please accept the software license agreement to proceed with the installation Copyright O 2013 Omixon Biocomputing Kft Omixon Target User Manual 600 Setup Omixon Target 1 4 0 License Agreement DY Please read the following important information before imni continuing Please read the following License Agreement You must accept the terms of this agreement before continuing with the installation SOFTWARE LICENSE AGREEMENT PLEASE READ THIS SOFTWARE LICENSE AGREEMENT CAREFULL
17. difficult to tell them apart and manual inspection of the results may be required e Pairs marked with a red pair flag have too many candidates in the list of possible pairs This maybe an indication of a problem in the sequencing either with contamination or demultiplexing e Pairs marked with a yellow pair flag have a few too many candidates in the list of possible pairs This is a warning flag HLA Typing Sample Result A more detailed view of the HLA Typing results for a single Sample Short reads are aligned to closely matching allele sequences then coverage is compared between allele candidates to get the best match es For each allele at each locus we supply e Detection Proportion of the allele with sufficient 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 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 marked as Best Match By clicking on the Analyse button next to the allele name the HLA Typing allele result screen is opened This page contains detail
18. discarded only a number matching your maximum coverage will be kept Low coverage threshold the threshold used for low coverage annotations in the Coverage annotation function e High coverage the threshold used for high coverage annotations in the Coverage annotation function Note that coverage annotation thresholds can be changed at any time on the Analyse sample page using the Coverage annotations function When the thresholds are changed the annotations are automatically recalibrated using the new values Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Variant Call 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 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 Variant Effect Prediction In the Pro version variant effect prediction can be run within OmixonTarget Variant annotations are generated using snpEff SNP Effect Predictor For HG19 chromosomes a variant effect database is provided for other references a custom database can be selected from the file system The most common codon sets are p
19. effect prediction can be run within Omixon Target Variant annotations are generated using snpEff SNP Effect Predictor For HG19 chromosomes a variant effect database is provided for other references a custom database can be selected from the file system The most common codon sets are provided and can be selected in the wizard Custom codon sets can be specified as well in an external file Advanced parameters can be chosen using a text file 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 browse that position within the Genome Browser which will also allow you to examine all the samples being viewed in the Samples Difference screen together Analysis Results Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 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 Varian
20. filter out this extra noise e Process all reads 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 e Read processing option You can now choose from a pre configured set of typical sequencing runs including Whole Genome and Whole Exome You can also choose Custom to manually set how many reads to process If you are not sure then use the All option to process all the reads e Maximum Reads Processed This is very important You may need to experiment with this value to find the best match for your data This dictates how many reads or how many pairs will be processed from each input file in order to do the HLA Typing For very targeted data sets including only the HLA loci this value doesn t usually need to be too high about 20 000 For larger kits such as the RainDance HLAseq kit the whole HLA superregion this will need to be significantly higher at least 2 000 000 Ignore Cross Mappings Some sources of data can be more noisy where there are reads from multiple loci that map to each other Generally the wider the sequencing the noisier the data This option allows reads that have been mapped to multiple HLA loci within the database to be ignored in the results and acts as a kind of noise filter e Ignore Rare Alleles There is now an option to ignore very rare alleles in the analysis they will not b
21. has sufficient coverage for typing percentage value Very uneven coverage will reduce the detection value Low detection scores will get a yellow warning flag Very low detection scores will get a red error flag Average coverage The average coverage across the whole allele High scores are usually better unless the coverage is very uneven i e lower detection score Warning error flags Flags might indicate an issue with the results See the Warning Flags page for more Filtering Alleles A summary of the currently active filters is displayed to the top right of the HLA results screens Locus filtering You can filter the displayed loci by using the Setup Loci function Status filtering By clicking the Assigned Only button unassigned allele candidates can be filtered out By clicking the Best Matches Only button lower ranked allele candidates can be filtered out Coverage filtering Using the Setup Filters function you can change the filtering options for the main candidates A minimum average coverage threshold can be set for the summary result table This limit can be set as an absolute coverage value i e the minimum coverage for the allele and or a relative coverage threshold minimum coverage percentage relative to the best matching allele The default value for minimum coverage is 10 for the absolute and 95 for the relative coverage filter function Alleles below the absolute coverage limit are shown as empty cell
22. 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 HLA 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 however this step can be re run at any time and will update the HLA database if a new version is available Once the Setup Typing function has been run the HLA Typing function becomes available From here multiple samples can be typed together within a single analysis It s possible to view the results 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 There are two display modes for the analyses the default card mode and searchable sortable list mode In the card display mode you can view the protocol set of parameters from any analysis run and save this protocol with a new name In the header section of the dashboard you can see the version and date of the IMGT HLA database that is currently configured together with the sequencers which have been set up within the tool Copyright 2013 Omixon Biocomputing Kft Omixon Target User Ma
23. known variants A Target is 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 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 Annotation Type Exon Gene Amplicon Press Next e Select a bed or gff file from the file system containing the annotations that describe your Target Press Finish After completing the wizard the chromosomes that are in the specified annotation files are downloaded automatically For each chromosome known variations and the snpEff database are also added Download problems If for some reason you cannot download the profile configuration file from within the tool e g due to network security 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 The setup files are packaged by chromosome and can be downloaded from the following links for example Chromosome 13 e http omixon download s3 amazonaws com target_ref_hg19_chr13 zip
24. least two similarly strong candidate pairs get an ambiguity flag purple This indicates that while these candidates are not 100 ambiguous i e with completely identical scores they are so close that it is difficult to tell them apart and manual inspection of the results may be required e Loci with more than 50 candidate allele pairs get an error flag red This may be an indication of a problem in the sample prep or sequencing either with contamination or demultiplexing e Loci with more than 30 candidate allele pairs get a warning flag yellow This is a warning flag Other indicators e Alleles displayed with a blue font are homozygous e Alleles displayed in italics are imbalanced i e present in a much smaller proportion of the reads that the main candidates HLA Genome Browser Introduction The HLA Genome Browser allows visual inspection of genomics data Multiple allele candidates can be browsed together The data items that can be visualised 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 insp
25. omixon target_windows x64_1_4 0 with_jre exe e 32 bit version named omixon target_windows_1_4_0_x86_with_jre exe We have tested Omixon Target with Windows 7 Windows XP and Windows 8 The 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 Installation steps Launch the executable After launching the installer Welcome dialog of the setup wizard appears 3 Setup Omixon Target 1 4 0 oa Welcome to the Omixon Target Setup Wizard This will install Omixon Target on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup Copyright O 2013 Omixon Biocomputing Kft Omixon Target User Manual Please accept the software license agreement to proceed with the installation License Agreement Please read the following important information before continuing Please read the following License Agreement You must accept the terms of this agreement SOFTWARE LICENSE AGREEMENT PLEASE READ THIS SOFTWARE LICENSE AGREEMENT CAREFULLY BEFORE DOWNLOADING OR USING THE SOFTWARE BY CLICKING ON THE ACCEPT BUTTON OPENING THE PACKAGE DOWNLOADING THE PRODUCT OR USING THE EQUIPMENT THAT CONTAINS THIS PRODUCT YOU ARE CONSENTING TO BE BOUND BY THIS AGREEMENT IF YOU
26. only in Target Pro edition The Map and Align step will always be performed The Variant Call and the Variant Effect Prediction steps are optional Both of these can be performed later using the Call Variants function or the mapped data can be copied elsewhere and other variant caller used instead The steps in this wizard Select Genomic Data Select Sequencer Advanced Options Variant Call Options Variant Effect Options 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 Map and Align Alignments can be run using Omixon s own aligner algorithm ORM or in the Pro version the BWA short read or long read algorithms 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
27. 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 e 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 This will start the Map and Align wizard You shouldn t need to change any settings in here you can review the settings 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 at least two new data items within the Sample one for the mapped data in BAM format and one for each reference contig 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 Analysis Dashboard select the Sample and then choose Browse Samples This will open the Genome Browser centered on the target you have defined in the Analysis Experiment You can start to analyse the var
28. 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 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 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 e General Properties e Choose Sequencer e Choose Target e Choose Species e Choose Genome e Choose Contig Once all these properties ar
29. using the Compare Samples function 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 fasta 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 function is only available in OmixonTarget Pro edition Steps in this wizard e Select Input File e Select P
30. 0 ace cosita a Casa le dea 51 1 5 5 HLA Typing Sample Result i sc0 20 24 ord erate oad ete e Ree iaa y ta ecb oneal 53 1 5 6 ALA Typing Allele Result gii creda een a e a AAA caca aid 54 1 5 7 Paired ResUl iia a id Ad a a de al 55 1 5 8 HLA Genome Browser ceci A ea a e 55 15 9 HLA Manage Tracks ironia cadrs eds A A r EA dea dd darian E 57 1 5 10 HLA EXpOrt Results comicos ci rat MEE tra dees anid Hodes Giese See isis 57 di LA Y BUG IMPOR a 57 1 512 ALA Export Table ii Ad E EE a A dl 57 1 5 13 HLA Typing Protocol Dashboard oooococococ eee 57 10 14 LA TYpINgiProtocol ds bl a dA Te W s Cera A e E tay Wace dd ac lena 58 Sandbox Browse gt tacto a A et abso over ed 58 16 4 Sandbox Genome BrowsSer scare eae a DAE aA a anaE a a Cased aj aed 58 16 2 Sandbox Open File esi a Vk Pad ea a FP dele ae WAU tebe dep Seana WebMasters 61 1 6 3 Sandbox Manage Files ccoo Set pia veel edi Sy ee eee ei ee We ee a 61 1 6 4 Sandbox Manage Sessions 0 ccc ete nee n tenn ene 61 136 5 Sandbox Manage Tracks carroceria 61 1 6 6 Sandbox Create Track coel bose eee e i etia dii peed e eke A diag A eee eee tlhe 62 16 7 Sandbox Add Track ni A a A wl anaes Sony anata Mae E E 62 SENS it A a hte bin A he ee i a A a el Sela ale lay 62 AV721SettingS DasnbOard nt sales Ne A tle A do le bee ater td did Meee A 62 1 22 Application Configuration ares cr heared tea 62 1 7 3 User Management i ees ek eet pe ce ee ea ene see de ee ele Che
31. 6 174 4 3 Create Track cuicos A ADE Be ee en ee RR AA 46 1 4 4 41AddsTrack a da o Lae Re Bice ler la bed O de tales 46 1 45 Import andiEXPOr sen o EA a aE bos 46 1 4 5 1 Import Reference Data ccc ce A A eee a e 46 1 4 5 2 Import Known Variant srna eedi See gia are ea alee daraa eee deere AA A E E a EAE E EEE aE i 46 1 4 5 3 Import Variant Effet i wesc te an oe ake a here acd do Tos 47 1 454 lmp AMMOTAL ONS e ier sce see e a E a hn peli a ps A aaar a a a a a E a a n o s Segall ce tna 47 1 4 5 5 Import Target Annotations ses cir Pee bana ia tabs locale wri beh AE AA a a E dae gle Poses a 47 1 4 5 6 Import Sequencing Data iisi cesa rn pee eee dh AE a E aA O a G EE 47 1 4 5 7 Import Mapped Data nani oi niie tarnai eee are E ATE AA dk E a da alee tc 47 1 4 5 8 Import Variant Calls yann ees teva a A a 48 1 4 5 9 Import Multiple Samples uti ia sos 48 1 4 5 10 Import Multiple Mapped 0 6 ccc eee nett eee e ee en ee eee teen eee anes 49 1 4 5 11 Export Mapped Data wou tee coat bebe a A ne Hae Ree ck ee 49 1 4 5 42 Export Mapped REGIONS si si lace menta ecatepec cl Saela had A cd cb aie eles 49 1 4 5 13Export Approved Variants ci A yale Sere ag lena a doch da eat ay 49 1 5 1 7 1 8 ALAS cited tr A A a oie Epa ate tng a aceite 49 1S SALA Typing Dashboard sri eagan a A als Salata Wapcalade Ad 49 1 5 2 HLATIDIN Setup EE A E A AE A eee rae ee 50 ELA A hyd eae a et wi ne ober a ey 50 1 5 4 HLA Typing Analysis R SUIt i 6
32. BIOCOMPUTING Y SOLUTIONS Omixon larget Designed with diagnostics in mind IS Omixon Target User Manual 1 Omixon Target User Manual veria a ea pe engl A 4 TARINTOUCION rd ala a a lira 4 SRA A OS 4 1 1 2 Sequencing Technologies ooocoococo eee e aana aaaea 4 VAS Modules ssc 05 5 a A niece oh ahs Matas ee Gael waned nia Mebane deep adds dia er dry 4 11 4 Key GOnCOpts cuota Ed ord arabia A San id aay Ele are oui 5 4 5 Bioinformatics NOTES remeare ea E sa e watt ballads De lela tee RA AAA 7 11 6 System requirement 3 0 42 a a A E See Oh ets Brae dd 7 11 7 Installation guide ci tee ea ed eae bee Pea A ew 8 1 1 7 1 Windows installation guide 2 0 0 ccc et eee tee eee rene eee nent ence eens 8 1 1 7 2 Mac OS X installation Quide a ot eroaa eves tt e daa Eee eve 13 TFA SaLINUe IMS tallat ON guide isisa lull cure axed atra O AO E dunt sadisne wus Ri 19 1 1 7 4 Pro Data Analysis Module installation notes 2 0 eee 25 1 1 7 5 Omixon Target Server Setup ete aaau aana 26 1 1 8 Acknowledgements c 5 ose se a orfeialie le tn A c 28 LL TUS a A RA Aide A tines bad AA es 29 SUSO a Al a au ica 29 1 2 1 1 Adding a Profile ici id A de A a nee Ee 29 1 2 1 2 Import sequencing fastq data 2 eee eee 30 1 2 1 3 Map the reads and call variants 2 0 nee eee 30 1 2 1 4 Results analysis and visualisation 2 2 0 0 00 eee 30 1 2 2 Custom Profile si cocos depend eeeet ra hin A epee hela ven meee lhe 31 12
33. C switches the browser between collapsed and uncollapsed mode T switches the browser between drag and drill navigation mode INSERT insert new annotation ENTER edit selected annotation DELETE delete selected annotation M move selected annotation to start at the locked cursor position R resize selected annotation to end at the locked cursor position Manage Tracks In this wizard you can hide and show any tracks and import create and delete additional annotation and variant tracks The visibility of any track can be set with the Show Track s and Hide Track s functions Hidden tracks are marked with a darker grey color in the Manage Tracks wizard track list Annotation tracks created by hand or added from file in this wizard are called User tracks These tracks can be edited within the Genome Browser Empty tracks can be created using the Create Track button These tracks are always editable Predefined i e non empty tracks can be imported from file using the Add Track button Annotations must be in gff or bed format variant calls are accepted in vcf format The editability of these tracks can be set during the import process For non empty tracks the type of the imported annotations variant exon gene or amplicon can be set as well The default annotation type is exon for gff and bed files and variant for vcf files Create Track In this wizard an empty annotation track can be created within Omixo
34. 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 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 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 Steps in this wizard e Select Input File e Select Reference Contigs e Select a Target Filter this will cause only data that overlaps your target region s to be imported Select maximum coverage i e choose how much coverage to import The maximum coverage feature should be used with car
35. Manager screen select Add New Server to setup client server connection Server manager f gt Add New Server import configuration Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Name your connection and enter exactly the same host and port settings which were set for server Add connection Connection name Omixon Target Server Server host target server mycompany com Server port 4380 Connection timeout ms 10000 Select server connection and do connect Server manager Omixon Target Server f Host target server mycompany Port 4380 Add New Server import configuration Connect IMPORTANT Check your firewall settings on the server computer Setup your firewall to let Omixon Target Server accept incoming connections The super user role The very first user on the server becomes a super user a server administrator who manages other user accounts For this reason the system administrator is supposed to register and login to Omixon Target at the setup period Thenceforward registration is not available any more and the login screen welcomes all connecting clients The clients user accounts are generated and given by the super user Exporting and importing connection configuration Instead of configuring the connection manually the users can choose to import a configuration file by clicking Import configuration button on Add New Server card in
36. OFTWARE BY CLICKING ON THE ACCEPT BUTTON OPENING THE y accept the agreement Ido not accept the agreement lt Back Next gt Cancel Select the path where you would like the application to be installed Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Setup Omixon Target 1 4 0 Select Destination Directory DY Where should Omixon Target be installed gt Select the Folder where you would like Omixon Target to be installed then click Next Destination directory Browse Required disk space 159 8 MB Free disk space 287 685 MB lt Back Next gt Cancel Select data directory where the permanent data files will be stored Setup Omixon Target 1 4 0 Data directory DY The directory where the application will store its Zz permanent data files lt This directory should be readable and writable by all users of the application It should have enough space to store large amount of genomic mapping and other data Browse lt Back Next gt Cancel Select temporary files directory Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Setup Omixon Target 1 4 0 Temp directory DY The directory where the application will store its temporary data files Z This directory should be readable and writable by all users of the application It should have enough space to store lar
37. Y BEFORE DOWNLOADING OR USING THE SOFTWARE BY CLICKING ON THE ACCEPT BUTTON OPENING THE PACKACE DOWNLOADING THE PRODUCT OR USING THE EQUIPMENT THAT CONTAINS THIS PRODUCT YOU ARE e accept the agreement y Edo not accept the agreement nstall4 lt Back Next gt Cancel Select the path where you would like the application to be installed eoo Setup Omixon Target 1 4 0 Select Destination Directory DY Where should Omixon Target be installed gt 9 Select the folder where you would like Omixon Target to be installed then click Next Destination directory Applications OmixonTarget Browse Required disk space 77 5 MB Free disk space 38 279 MB lt Back Next gt Cancel Select data directory where the permanent data files will be stored Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 600 Setup Omixon Target 1 4 0 Data directory DY The directory where the application will store its permanent 1 data files A This directory should be readable and writable by all users of the application It should have enough space to store large amount of genomic mapping and other data Users omixontargetuser ogve Browse lt Back Next gt Cancel Select temporary files directory 600 Setup Omixon Target 1 4 0 Temp directory DY The directory where the application will store its temporary y data files ty This directory should be readable and writ
38. 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 http www ncbi nlm nih gov SNP 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 Map and Align Samples Introduction Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 l
39. able by all users of the application It should have enough space to store large amount of genomic mapping and other data lt Back Next gt Cancel After clicking the Next button the installation process starts When it is finished the Completing the Omixon Target Setup dialog indicates the success of the installation Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 000 Setup Omixon Target 1 4 0 Completing the Omixon Target Setup Wizard Setup has finished installing Omixon Target on your computer The application may be launched by selecting the installed icons Click Finish to exit Setup Finish In Application list Omixon Target is available now By clicking the Omixon Target app icon application starts and is ready for use e00 3 OmixonTarget SiO A a Arrange Share Path __ New Folder _ Delete Get Info Search A qn FAVORITES Applications E All My Files CON AirDrop a Ys J Applications ci Omixon Target omixon target app ha Documents Uninstaller app Downloads FERE L Movies JJ Music o 7 B Pictures E a Sm aw a a omixontargetuser clientlib doc GATK lib Uninstalling the product If you want to remove Omixon Target run the uninstaller app which is available in the directory where the application was installed If you intend to use future version of Omixon Target and you don t want your work data to be deleted keep the check
40. aired Read Options e 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 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 Import Multiple Mapped This function is available from the Analysis Dashboard If you have already mapped some short read data you can import it with this function and then run a variant call in order to identify the variants It is essentially identical to the Import Mapped Data func
41. all be selected together in the Select input files page Ensure that the Paired read import and Automatic pair selection checkboxes are selected in the Paired read options page If there is a problem with the automatic pair selection you will have to manually tell the tool which is the first paired file and which is the second In this case select the first file for each sample in the Select input files page you can still select multiple files for multiple samples and then deselect the Automatic pair selection checkbox and choose the second file for each sample in the Paired read options page 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 Paired read options Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual For most Illumina data sets the defaults will be fine 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 Options are FR forward reverse RF reverse forward and FF forward forward Advanced Options These options dictate 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
42. and import all the data required for each Profile Appendix Keyboard shortcuts The following generic keyboard shortcuts are available in Omixon Target e F1 key opens the Help for the current page or wizard F8 key closes the window and exits the application 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 e ALT F4 closes Omixon Target CTRL C V X copies pastes deletes text in text boxes SHIFT CTRL left click can be used for multiple selection deselection Copyright 2013 Omixon Biocomputing Kft
43. anual 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 any other 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 is now available in the Pro Module Variant Calling Tools These tools are only available in the optional Data Analysis Modules Standard and Pro Omixon currently uses 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 on 3 platforms e MacOSX e Linux e Windows The recommended hard
44. arget 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 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 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 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
45. 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 Experiment 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 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 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 Analysis References Configure References for an Analysis Omixon Target has
46. box unchecked If you wish to cleanup the working directory then selet the option Yes Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 800 OmixonTarget 1 4 0 Uninstall _ Select Additional Tasks PNT Which additional tasks should be performed l Select the additional tasks you would like Setup to perform while uninstalling Omixon Target then click Next Yes Do you want to remove your working data install4j Next gt Cancel Click Next to proceed with the uninstallation process e00 Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Are you sure you want to completely remove Omixon Target and all of its components Click Next to continue or Cancel to exit Setup Next gt Cancel Finally the installer informs you about the outcome of uninstall Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 600 Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Omixon Target was successfully removed from your computer Finish Linux installation guide We provide two versions of installer package for Linux operating systems both are bundled with a Java Runtime Environment JRE e 64 bit version named omixon target_unix_1_4 0 with_jre sh e 32 bit version named omixon target_unix_1_4 0_x86_with_jre sh The install packages are single file shell scripts suitable to install on various Linux distributions The 32 bit o
47. browsed together Analyse Sample 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 function also generates a target based coverage report containing some annotations e g for zero coverage regions within the defined target and basic coverage statistics 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 On this screen variants for all samples are shown within a single table The selected samples can also be browsed together and compared visually in the Genome Browser Manual Approval 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 the Automatic Approval or Automatic Rejection functions or via a m
48. chemes 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 allows unlimited HLA Typing Please contact sales omixon com for a quote 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 The section covers the main concepts for the Data Analysis modules Target Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Data Analysis 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 samples that are sequenced there is a simple data management hierarchy in the tool The sequencing data will belong to a
49. ck track name i e the name shown in the Browser track header can be specified For vef files the track type is automatically set to Variant annotation The editability of the imported track can be set as well Note that editing is only supported for bed formatted files at the moment Settings 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 i e this will delete all imported and analysed data and reset the whole of Omixon Target back to a brand new status This function is essentially only recommended for demonstrations or if you really do want to start again from scratch Application Configuration This wizard allows you to set up a mail server via SMTP for sending bug and error reports directly to Omixon Settings selected in the Application Configuration wizard are saved and will still be in effect after Omixon Target is restarted 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 Genome Browser Setup You can
50. croll through the reads at the current position area PAGE UP PAGE DOWN scroll alongside the reference contig in bigger steps HOME END jump to the beginning end of the current reference contig zoom in and out SPACE locks unlocks the cursor i e green line when unlocked red line when locked BACKSPACE shows the whole reference contig i e zooms out totally or when the cursor is locked jumps to the locked cursor Mousewheel zoom in out in Drag mode or move alongside the reference in Drill mode SHIFT mousewheel scroll through the reads at the current position area Left mouse button can be used for dragging the display in Drag mode or for highlighting and zooming in on a region in Drill mode by clicking and releasing the left button e TAB SHIFT TAB jump to next previous reference contig Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual e CTRL J opens the Jump to position wizard Here you can specify a reference contig and a coordinate or area on that contig and jump to the selected location e CTRL F opens the Jump to annotation wizard Here you can specify the name or partial name of an annotation e g gene amplicon exon and jump to the selected annotation e CTRL S opens the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible reference contig and then step through the hit positions CTRL M enter f
51. ct a snpEff database bin file Press Finish Reference Genome Dashboard Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 bed or gff 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 need to worry about this function 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 impo
52. d custom candidate you can add any alleles of the currently selected gene from hla dat Reads that can be aligned to this custom allele will be shown in the browser Note that allele candidates that are already in the candidate list for that gene cannot be added again Remove custom candidate the previously added custom allele can be removed by this function Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Shortcuts Keyboard mouse navigation options and shortcuts UP DOWN arrow keys scroll alongside the reference contig LEFT RIGHT arrow keys scroll through the reads at the current position area PAGE UP PAGE DOWN scroll alongside the reference contig in bigger steps HOME END jump to the beginning end of the current reference contig zoom in and out SPACE locks unlocks the cursor i e green line when unlocked red line when locked BACKSPACE shows the whole reference contig i e zooms out totally or when the cursor is locked jumps to the locked cursor Mousewheel zoom in out in Drag mode or move alongside the reference in Drill mode SHIFT mousewheel scroll through the reads at the current position area Left mouse button can be used for dragging the display in Drag mode or for highlighting and zooming in on a region in Drill mode by clicking and releasing the left button e CTRL J opens the Jump to position wizard Here you can specify a reference contig and a coordina
53. define the starting i e used when the software is freshly started graphical and navigation settings for the Genome Browser in the Data Analysis module the free Sandbox Genome Browser and the HLA Browser here These settings can be saved and will still be in effect after Omixon Target is restarted The genome browser settings are user specific Display configuration On this screen the orientation of the browser screen horizontal or vertical the navigation mode Drag amp Zoom or Drill amp Scroll Color setup On this screen the color codes for nucleotides used in the genome browser can be changed Data options In this part of the wizard you can set whether certain groups of variants should be shown or hidden based on variant status accepted rejected pending or all or variant type insert or deletion Soft clips can be shown or hidden as well Advanced settings 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 default setting is 20 The minimum number of pixels for collapsed read width can be set as well Tip For further details about the Display and Data settings see the Genome Browser Sandbox Genome Browser or HLA Browser help pages Upload license Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual You can upload a license with this wizard Just select the fil
54. download s3 amazonaws com target_brca_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 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 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 or sff file or a pair of files for paired data from the file
55. e it will take the first X reads pairs it finds it s not a sampling method 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 another 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 e Select Annotation Type e Select Input Files Import Options e 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
56. e detection facility and will not normally be needed It may be useful in detecting contamination across multiple samples or helping to diagnose very noisy data Assigning Alleles For each allele one or more allele candidates can be assigned There are four different ways to assign allele candidates e Candidates can be assigned manually by clicking on the check mark before the allele candidate s name e All best matching candidates i e the best allele candidates based on coverage and detection statistics can be assigned using the Assign Best Matches button e All unambiguous results can be assigned using the Assign Unambiguous button All allele candidates can be assigned using the Assign All button All assignments can be deleted by the Unassign All button Note that all the Assign buttons on this screen affect all the displayed samples and alleles they don t affect alleles that are not being displayed due to filtering criteria If you prefer to do the assignments separately for each sample allele you can find similar functions by using the Sample Details and Allele Details buttons Warning Flags e Alleles displayed with the blue font are homozygous e Alleles displayed in italics are imbalanced i e present in a much smaller proportion of the reads that the main candidates Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Results are annotated with warning flags Some of these flags apply t
57. e included or reported in this case If this option is not chosen then these rare alleles will be marked in the results If this option is greyed out you will need to Setup HLA Typing again in order to import the rare alleles database e Typing precision When 8 digit typing is set and both the sequencing and the database have the intronic information then Omixon Target tries to give an 8 digit result Otherwise if falls back to 6 digit e Save read mappings bam format Use this setting to save the mapped reads into a bam format if you would like to browse them in the HLA Genome Browser HLA Typing Analysis Result Introduction High level overview of the HLA Typing results A high level overview table of the HLA Typing results for one or more submissions of the HLA Typing tool This can include the summary results for many samples You can use Ctrl mouse wheel to zoom in and out of the table Short reads are aligned to closely matching allele sequences then coverage is compared between allele candidates to get the best match es The algorithm will automatically select two or more best matching alleles for each locus These can be manually overridden assigned by the user Each Sample can be selected and the following options are available Paired Results all possible pairs of candidates for each locus select pairs together e Sample Details the detailed results for a sample e Allele Details the detailed results for an
58. e is provided for other references a custom database can be selected from the file system The most common codon sets are provided and can be selected in the wizard Custom codon sets can be specified as well in an external file Advanced parameters can be chosen using a text file Analyse Sample Introduction 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 It s possible to start the Approval Dashboard from here in order to approve the variants ready for further analysis and reject any low Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual quality or off target variants that should be ignored It s also possible to navigate to the Report Dashboard to see graphs charts and other summary information about the Sample data currently under development The Coverage report can also be reached from this screen This report contains statistics about the coverage of the sample A coverage based annotation tool is also available from here Summary On the Summary tab a short list of basic information about the selected sample is shown Name of the fil
59. e set for an Experiment there will be automatically used by Analyses that belong to the Experiment 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 http www ncbi nlm nih gov SNP Experiment Targets Configure Targets for an Experiment 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 Alignment and Variant Call Analysis Dashboard The main purpose
60. e that contains the variant call results e Name of the file that contains the expected variants Name of the currently selected contig Tip you can chose a different contig using the Switch contig button Total number of variants found on the current contig Total number of variants found on all the contigs On target Variants On this tab all variants located in the specified target are listed you can check the targets on the Analysis targets screens For each variant the following fields are always available e Start pos the start position of the variant e End pos the end position of the variant e Ref value the reference bases at the location of the variants Type the type of the variant SNP or INDEL e Value alternative value i e the nucleotide s supported by the reads e Frequency frequency of the alternative allele in the reads e Quality quality of the variant e Coverage coverage at the position of the variant e Id doSNP ID of known variants e Status status of the variant possible values Pending Approved and Rejected Note that when the variant effect prediction function is run additional fields are shown between Coverage and Id For a description of these additional fields see the Variant effect results section of this page Off target Variants On this tab all variants located outside the specified target are listed you can check the targets on the Analysis targets scr
61. e 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 HLA Typing Export Log This function will export the Omixon Target log file This is useful if you have encountered a problem within the tool and would like to notify Omixon support staff of the issue they will usually ask for a log file so that they can investigate your issue and ensure that it is resolved as soon as possible Export Audit Log This function exports the full audit log of everything that has happened to each and every sample inside Omixon Target This is useful for audit purposes 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 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 next recommended step is to use Add Profile to add one or more of the pre configured Profiles to the tool which will download
62. eating a startup icon in the system menu this symlink is optional for most users 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 these tools manually Once they are on the PATH they can be used General notes and configuration In case you have all the tools installed already and they are available on your PATH then no additional configuration is needed Otherwise 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 natives etc folder of your installation directory 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 Below 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 manager on Debian based systems apt get install samtools Bowtie Homepage of the tool http bowtie bio sourceforge net Version used for testing 0 12 8 Copyright 2013 Omixon Biocomputing Kft Omixon Target U
63. ected 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 Settings and Functions 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 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 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
64. ed Modifier e START_GAINED Start gained Low e SPLICE_SITE_ACCEPTOR Splice acceptor High e SPLICE_SITE_DONOR Splice donor High e START_LOST Start lost High e SYNONYMOUS_START Syn start Low e CDS Cds Modifier e GENE Gene Modifier e TRANSCRIPT Transcript Modifier e EXON Exon Modifier e EXON_DELETED Exon deleted High e NON_SYNONYMOUS_CODING Non syn coding Moderate e SYNONYMOUS_CODING Syn coding Low e FRAME_SHIFT Frame shift High Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual CODON_CHANGE Codon change Moderate CODON_INSERTION Codon insert Moderate CODON_CHANGE_PLUS_CODON_INSERTION Codon ch ins Moderate CODON_DELETION Codon delete Moderate CODON_CHANGE_PLUS_CODON_DELETION Codon ch del Moderate STOP_GAINED Stop gained High SYNONYMOUS_STOP Syn stop Low STOP_LOST Stop lost High INTRON Intron Modifier UTR_3_PRIME Utr3 prime Moderate UTR_3_DELETED Utr3 deleted Moderate DOWNSTREAM Downstream Modifier INTRON_CONSERVED Intron cons Modifier INTERGENIC_CONSERVED Inter cons Modifier INTRAGENIC Intragenic Modifier RARE_AMINO_ACID Rare amino acid High NON_SYNONYMOUS_ START Non syn start Low Coverage Annotations The coverage report contains some custom annotations for target regions where the coverage is too high too low or zero These can be regenerated using different threshold values
65. ed 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 best matching candidates i e the best allele candidates based on coverage statistics can be assigned using the Assign Best Matches 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 assignments separately for each sample allele you can find similar functions on the HLA Typing sample and allele result screens The
66. eens Fora short description of the fields shown in the table see the On target Variants section Variant effect results Variant effect prediction results are generated using snpEff Pro Server version only These results are shown on the Analyse sample page in the On target variants and Off target variants tables The snpEff result fields are the following e Effect effect of the variant Impact estimated impact of the variant possible values are Modifier Low Moderate or High The impact is estimated based on the type of the effect For details about impact categories see the table below e Region the coding region where the variant is located in according to the SnpEff database e Codon the codon change caused by the variant reference codon alternative codon the variant is shown in upper case the rest of the codon is shown in lower case e Aa amino acid change One letter code of the reference amino acid followed by the location of the amino acid followed by the one letter code of the alternative amino acid If the mutation is synonymous the second letter is missing The original snpEff results are shortened to fit into the variant call table For a full list of the abbreviations used see the list below Original result Omixon Target Result Estimated impact e INTERGENIC Intergenic Modifier e UPSTREAM Upstream Modifier e UTR_5_PRIME Utr5 prime Modifier e UTR_5_DELETED Utr5 delet
67. ele He beta eee eee ele 62 1 7 4 Genome Browser Setup 0 0 a a E a E EAE E ene e EE E r teen eet eee 62 12725 Upload license at A A A va eka eat A dene Hoek E Bataan O 62 AL SEXO LOGS ot ta a Real aroma Sty cata lpn ete ale e het tate Sahn gale i ep aN nee eee bd cdo tea 63 17r Export AUGItiLOG Disco ie A eek acho RA angen eer Maden ee ake Bod chug foe 63 1 7 8 ResetEverything zei ccc sate eee oe ce eee haa Aa ee ee glee ioe ely ee Leta cea DRE ee eel 63 A b GaP RRS Resa A RAN 63 1 821 Keyboard shortcuts ata A are esa AS ates Oe area WA tn deer bare aren ett 63 Omixon Target User Manual Omixon Target User Manual Introduction Introduction Omixon Target the tool is a suite of software for analysing targeted sequencing 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 www omixon com omixon target quick start guide 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 resu
68. ement functions as well as a full featured NGS Genome Browser This module is available free of charge The Sandbox Genome Browser Module works on Windows Linux and Mac OS X Standard Data Analysis Module Optional Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 variant effect prediction an approval work flow for automated or manual filtering and approval of variants and an automated coverage analysis and coverage annotation pipeline The Standard Module works on Windows 64 bit recommended Linux 64 bit recommended and Mac OS X Alignment There is one short read aligner available Omixon s own ORM Omixon Read Mapper Variant Calling The variant calling is provided by a GATK pipeline using the Broad Institute GATK variant caller This gives excellent results with Illumina data Variant Effect Prediction There is the option to import an SnpEff database for variant effect prediction This will be done automatically for HG19 if any of the built in profiles BRCA CFTR are used or if any profiles are created for HG19 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
69. enes or Exons Target configuration via Targets lists and Target Dashboard e Create Target e Configure Target Adjusting Memory Usage 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 is enough However it might not be sufficient for the underlying alignment algorithms to run If you get Out of Memory or GC Limit Exceeded errors then you will need to either increase the amount of memory available to Omixon Target or run the tool with reduced sensitivity The first option is 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 there is a file called omixon target vmoptions on the Mac it is found in Applications OmixonTarget app Contents Info plist Open this file to modify a single VM virtual machine option Xmx5g Replace original to the following 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
70. faa nA a oleae ii 37 1 4 3 Alignment and Variant Call 2 0 ett tent t nee ee 38 1 4 3 1 Analysis Dashboard x a rrii se ca ee e eee A EE eee a ee he 38 143 2 DeSIGM ANALYSIS vii iii A SES ee Bek Bea A be ae 1 ae OA ti 38 1 4 3 3 Analysis sREIGrenCes 154 6 nee i ote teeter a A de Venere Dl tae 38 VA Si4 Analysis Var Gets jes sc iia a a a a amada adi 38 1 4 3 5 Map and Align Samples a eee eet tenet eet nee 38 1 4 3 6 Sample Difference sc ve e ccna See pe eee eee ey eee ee a ee ee eee ny 39 1 4 3 7 Analysis Results onaren e ti eben FS ee de oa aire Seep i hae Ta na RE ee Ed ec eee 39 1 4 3 8 Sample Dashboard syy scout n a aa eee adnan fee A eee Be eed 40 1 4 3 9 Create Sample ia a sates a add aid 40 1 4 3 10 Map NAM iia Ad A da ate one p E 40 1 4 3 4 1 Call Variants is a ro n a a ea heed RADA LADA nae tl ee E eee ey 41 1 4312 Analyse Sample aunando o ie e al Mead et o e de lo el O de eel 41 1 4 3 13 Coverage Annotations oooocococco ete n ttn e teens 43 1 4 3 14 Approval D shbdar dicta pain lc Soe cd la te fe passat goR Ub bs waa pcp ef win ace bal ae EAE EA ested aes ae 43 1 4 3 15AUtO Approve Snine ar RE Pa A A a Ada cda 43 1 4 3 16 Auto Reject oa eos ei A OO EDU ea ee eRe Dee cea A 43 LASA LUTO OSO a s Wipe ile ella ic salts tad 43 1 4 4 Data Analysis Module Browser 0 0 cette eee 43 1 4 4 1 Genome Browser isco se pea neha ha a chee ee eal a Debate ee ed eke 43 14 42 Manage Tracks viii a A Shee a dc 4
71. for the high and low coverage limits i e target regions with higher coverage than the High coverage threshold and target regions with lower coverage than the Low coverage threshold will have new annotations created Any existing high low coverage annotations will be discarded 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 Some 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 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 seque
72. g 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 copy the reference sequence belonging to a selected item read or annotation by clicking on the Copy reference to clipboard button 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 Context menu By clicking the right mouse button you can open the Context menu In the HLA Browser this menu contains the following functions e Search sequence with this function you can search for a specific nucleotide sequence in the reference of all visible alleles e Toggle fullscreen you can toggle between full screen i e only the browser is shown and the normal view e Toggle reference masked with this function you can hide all the bases shared by the different allele reference sequences on the screen This way only the differences between the allele candidates are shown e Ad
73. ge amount of genomic mapping and other data Browse lt Back Next gt Cancel Set symlink location It is useful for users who 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 Setup Omixon Target 1 4 0 Select Directory for Symlinks DY Where should Omixon Target create symlinks to the gt executables Ey Select the Folder where you would like Omixon Target to create symlinks then click Next Create symlinks Destination directory usr local bin Browse lt Back Next gt Cancel After clicking the Next button the installation process starts When it is finished the Completing the Omixon Target Setup dialog indicates the success of the installation Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual x Setup Omixon Target 1 4 0 Completing the Omixon Target Setup Wizard Setup has finished installing Omixon Target on your computer The application may be launched by executing the installed start scripts Click Finish to exit Setup Finish An application icon for Omixon Target is placed into the System menu By clicking on this icon the Omixon Target application starts and is ready for use Applications Places amp Accessories a Education oP Games Graphics Internet Office Programming install4j 5 IDE System Tools gt
74. genomes can be added to 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 or sff 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 e Finally variant call results can be imported alone without any short read data at all and visualized and analysed 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 Sample Dashboard approva
75. ghter colour The attributes of the annotation element can be edited using the Edit selected annotation function this button is located on the middle bottom panel next to the Create new annotation button You can also open the annotation editor screen by pressing the ENTER button after selecting an annotation e To resize an annotation entry in the browser select the annotation lock the cursor you can do this by pressing Space on your keyboard then press the Resize selected annotation button on the middle bottom panel or press R on the keyboard The selected annotation will be resized to end at the cursor position e To move an annotation entry in the browser select the annotation lock the cursor you can do this by pressing Space on your keyboard the press the Move selected annotation button on the middle bottom panel or press M on the keyboard The selected annotation will be moved to the cursor position Deleting tracks To delete an annotation entry select the annotation then press the Delete selected annotation button red X symbol on the middle bottom panel or the Delete button on your keyboard Showing or hiding tracks All tracks both built in and user created tracks can be temporarily hidden using the Hide track s button in the Track Manager wizard Navigation in the Browser Zooming By default the Genome Browser starts in Drag mode where the mouse wheel or and keys can be used t
76. iants From the Sample Dashboard select Analyse sample 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 Browse Feature which will switch to 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 this with an example In this example we will create the profile for one of the ready to use lon AmpliSeq panels lon Am
77. in tracks these tracks are automatically imported into the Omixon Target database and are not editable Sandbox Manage Files On this screen you can manage files selected in the Open wizard Both reference files reference contigs and annotations and sample files alignments and variant calls can be deleted individually The order of sample files can be changed as well by selecting a single alignment or variant call and moving it up or down in the list You can also create new sessions using the Save as function This will basically create a duplicate of the current session including the current browsing location with a new name This function can be used for saving interesting or problematic regions for further analysis or simply for archival reasons Tip For temporarily hiding tracks see the Manage Tracks function Sandbox Manage Sessions In this wizard you can create new empty sessions and delete copy rename or open existing sessions The current session is not deletable and at least one session should exist at all times this means that if you have a single session this session can not be deleted Tip The current session is marked with a yellow frame Sandbox Manage Tracks In this wizard you can hide and show any tracks and import create and delete additional annotation and variant tracks The visibility of any track can be set with the Show Track s and Hide Track s functions Hidden tracks are
78. is will automatically be created for the imported results Note that these files don t contain alignment information so you won t be able to use the HLA Genome Browser for these samples HLA Export Table Every HLA typing result table can be exported from Omixon Target in either csv txt tab delimited text xls or xlsx format Note that only visible results are exported i e if an allele is filtered out based on low coverage it won t be shown in the exported file either There are several similar export table functions in the HLA module of Omixon Target e On the HLA Typing dashboard you can export an overview of all analyses Note that the Table Export function is only available when the Display mode is set to table e On the HLA Typing analysis results dashboard you can export all the results from an analysis If you select the Overview export only the allele names will be exported The Detailed table also contains flags and statistics for each allele candidate e On the HLA Typing sample result page you can export data for a single allele by pressing the small Export button in the allele header e On the HLA Typing allele result page you can export detailed statistics of the allele candidates of a single allele HLA Typing Protocol Dashboard Introduction This function is available from the main HLA Typing Dashboard You can view edit and use protocols from here Protocols are stored sets of HLA Typing analy
79. ist 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 e Map and Align using Omixon mapper or BWA e Variant Call using GATK or Samtools optional e Variant Effect Prediction using snpEff optional The Map and Align step will always be performed The Variant Call and Variant Effect Prediction steps are optional The Variant Call and Variant Effect Prediction steps can be performed later using the Call Variants function or the mapped data can be copied elsewhere and other variant caller used instead The steps in this wizard e Select Reference Data e Select Sequencer e Advanced Options e Variant Call Options e Variant Effect Options 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 Map and Align Alignments can be run using Omixon s own aligner algorithm ORM or in the Pro version the BWA short read or long read algorithms 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 sectio
80. ith 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 will be downloaded and imported automatically The Create Custom Profile makes it easy 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 References and Targets 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 BRCA chr13 and chr17 e CFTR chr7 The two pre configu
81. jump back to that feature at any time by using the small arrow button at the bottom of the display Search for a nucleotide sequence By pressing CTRL S you can open the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible allele sequences and then step through the hit positions 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 candidates can be hidden By clicking the Best Matches Only button lower ranked allele candidates can be hidden Analysing result By clickin
82. l 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Genome Browser There is a genome browser included in the tool This allows you to browse 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
83. les 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 Select the Import known variants item in the Actions menu on the left e 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 of 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 Import Target You should start at the Data Analysis Dashboard Click on Targets and choose the Import Target button from the menu above the list of Targets Give your new Target a name e g Custom Cancer Gene Panel Select the Annotation Type Amplicon Select the bed file for your target regions from the file system Choose the Reference your new custom reference Select the Contigs chromosomes Press Finish This will start a new background task Once this task has completed you can create a new Experiment and
84. lts Data Analysis Omixon Target includes preconfigured support for analysing some of the most common human diagnostic targets such as 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 sample 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 Modules Omixon Target has a modular structure Omixon Target consists of a free 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 GFF BED formats Free Sandbox Genome Browser Module This is always included as standard with Omixon Target It includes some data manag
85. marked with a darker grey color in the Manage Tracks wizard track list Annotation tracks created by hand or added from file in this wizard are called User tracks These tracks can be edited within the Genome Browser Empty tracks can be created using the Create Track button These tracks are always editable Predefined i e non empty tracks can be imported from file using the Add Track button Annotations must be in gff or bed format variant calls are accepted in vcf format The editability of these tracks can be set during the import process For non empty tracks the type of the imported annotations variant exon gene or amplicon can be set as well The default annotation type is exon for gff and Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual bed files and variant for vcf files Sandbox Create Track In this wizard an empty annotation track can be created within Omixon Target The name and type of the annotation can be set the annotation type can be exon gene or amplicon This user defined annotation track is stored in a bed formatted file at a location selected by the user When the annotation track is edited within the Genome Browser this storing bed file is automatically updated and saved Sandbox Add Track In this wizard annotation tracks in vcf gff or bed format can be imported from external files The type of the imported track variant exon gene or amplicon and the Fallba
86. mputing Kft Omixon Target User Manual HOME END jump to the beginning end of the current reference contig zoom in and out SPACE locks unlocks the cursor i e green line when unlocked red line when locked BACKSPACE shows the whole reference contig i e zooms out totally or when the cursor is locked jumps to the locked cursor Mousewheel zoom in out in Drag mode or move alongside the reference in Drill mode TAB SHIFT TAB jump to next previous reference contig SHIFT mousewheel scroll through the reads at the current position area Left mouse button can be used for dragging the display in Drag mode or for highlighting and zooming in on a region in Drill mode by clicking and releasing the left button e CTRL J opens the Jump to position wizard Here you can specify a reference contig and a coordinate or area on that contig and jump to the selected location e CTRL F opens the Jump to annotation wizard Here you can specify the name or partial name of an annotation e g gene amplicon exon and jump to the selected annotation e CTRL S opens the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible reference contig and then step through the hit positions CTRL M enter full screen mode hide all menus and show the browser region in full screen ESCAPE exit full screen mode X switches the browser between horizontal and vertical mode
87. multiple items Press Next Select Annotation Type Amplicon Press Next Select the AmpliSeqCancerAmplicons bed file from the file system Leave the next step set to Download file s 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 HG19 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 you would like to download Option 2 Using the Create Custom Profile function If you would like t
88. n Target The name and type of the annotation can be set the annotation type can be exon gene or amplicon This user defined annotation track is stored in a bed formatted file at a location selected by the user When the annotation track is edited within the Genome Browser this storing bed file is automatically updated and saved Add Track In this wizard annotation tracks in vcf gff or bed format can be imported from external files The type of the imported track variant exon gene or amplicon and the Fallback track name i e the name shown in the Browser track header can be specified For vef files the track type is automatically set to Variant annotation The editability of the imported track can be set as well Note that editing is only supported for bed formatted files at the moment Import and Export 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 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
89. n 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 e Low coverage threshold the threshold used for low coverage annotations in the Coverage annotation function e High coverage the threshold used for high coverage annotations in the Coverage annotation function Note that coverage annotation thresholds can be changed at any time on the Analyse sample page using the Coverage annotations function When the thresholds are changed the annotations are automatically recalibrated using the new values Variant Call 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 e 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 Pro version of Omixon Target a Samtools variant call is also available from this wizard Samtools is the suggested variant caller for long reads lon Torrent and 454 Variant Effect Prediction In the Pro version variant
90. nager wizard Navigation in the Browser 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 Jump to a position You can jump to a position in the reference using Jump To Position or jump to one of the annotations within the Target using Jump To Target or Jump to annotation 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 The Jump To Position wizard can be opened by pressing the CTRL and J keys on the keyboard After the wizard is opened you can type in a single coordinate or a pair of coordinates and you can specify a reference contig When a reference contig is not chosen the currently shown contig is used Coordinates can be specified with or without thousand delimiters i e 15200 15 200 and 15 200 are all accepted and mean the same Some examples the currently selected reference contig is chr1 e CTRL J 15200 ENTER gt The Genome Browser display will jump to chr1 15200 e CTRL J 15 200 15 400 ENTER gt The Genome Browser display will show the area chr1 15200
91. nates and you can specify a reference contig When a reference contig is not chosen the currently shown contig is used Coordinates can be specified with or without thousand delimiters i e 15200 15 200 and 15 200 are all accepted and mean the same thing e CTRL J chr2 15 200 ENTER gt The Genome Browser display will jump to chr2 15200 Jump to an annotation The Jump To Annotation wizard can be opened by pressing the CTRL and F keys on the keyboard After the wizard is opened you can type in the name or the beginning of the name of an annotation e g gene exon amplicon When the specified search term is ambiguous i e more than one annotation name starts with the specified string a list of annotations is presented and the desired annotation can be selected from the list The input field in this wizard is not case sensitive i e brcal and BRCA1 are both accepted and mean the same thing Search for a nucleotide sequence By pressing CTRL S you can open the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible reference contig and then step through the hit positions Shortcuts Keyboard mouse navigation options and shortcuts e UP DOWN arrow keys scroll alongside the reference contig e LEFT RIGHT arrow keys scroll through the reads at the current position area e PAGE UP PAGE DOWN scroll alongside the reference contig in bigger steps Copyright 2013 Omixon Bioco
92. ncing 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 Data Analysis Module Browser Genome Browser Introduction Multiple Samples can be browsed together The data items that can be visualised include Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual e Reference Sequence e Gene Annotations e Exon Annotations e Amplicon Annotations And for each Sample Actual Variant Annotations Expected Variant Annotations Short Reads including Coverage Low High Zero coverage annotations 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 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
93. next to a homozygous result indicates a possible imbalanced allele in the result This is a warning that there may be an issue with the primer and one of the two candidate alleles is present in a very low proportion of the read data compared to the other i e imbalanced result Using the imbalanced filter these imbalanced candidates can be viewed Paired candidate flags e Where there are multiple allele candidate pairs with very similar coverage and detection statistics these will be marked with a purple pair flag This indicates that while these candidates are not 100 ambiguous i e identical scores they are so close that it is difficult to tell them apart and manual inspection of the results may be required e Pairs marked with a red pair flag have too many candidates in the list of possible pairs This maybe an indication of a problem in the sequencing either with contamination or demultiplexing e Pairs marked with a yellow pair flag have a few too many candidates in the list of possible pairs This is a warning flag HLA Typing Allele Result Allele candidate level HLA typing results A more detailed view of the HLA Typing results for a single Allele candidate Short reads are aligned to closely matching allele sequences then coverage is compared between allele candidates to get the best match es For each exon of each allele candidate we supply e Detection Proportion of the exon with sufficient coverage High values a
94. nments variant calls and annotation files can be imported within a single wizard Multiple browsing sessions i e basically sets of tracks with visual settings can be saved Saved sessions can be reopened with a few clicks and can be edited freely at any time The last used browsing session is stored and reopened Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual automatically when Omixon Target is restarted The browser comes with a built in HG19 reference so if the selected alignment annotation or variant call files are HG19 based the required reference chromosomes are automatically detected and loaded Settings and Functions 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 Filtering the Short Reads You can display more short reads by using the collapsed pile up view In collapsed mode 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
95. nored option during the analysis then they will be marked with a red exclamation mark in the results e A yellow balance icon next to a homozygous result indicates a possible imbalanced allele in the result This is a warning that there may be an issue with the primer and one of the two candidate alleles is present in a very low proportion of the read data compared to the other i e imbalanced result Using the imbalanced filter these imbalanced candidates can be viewed Paired candidate flags e Where there are multiple allele candidate pairs with very similar coverage and detection statistics these will be marked with a purple pair flag This indicates that while these candidates are not 100 ambiguous i e identical scores they are so close that it is difficult to tell them apart and manual inspection of the results may be required e Pairs marked with a red pair flag have too many candidates in the list of possible pairs This maybe an indication of a problem in the sequencing either with contamination or demultiplexing e Pairs marked with a yellow pair flag have a few too many candidates in the list of possible pairs This is a warning flag Paired Result In this screen you can view and assign HLA typing results by seeing the most likely pairs of alleles per locus This screen shows the top pairs of allele candidates and allows one or more pairs to be selected together Results are annotated with warning flags e Loci with at
96. nual 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 you 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 http omixon download s3 amazonaws com target_haplotype_db zip HLA Typing Introduction 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 files for the input You also need to select which sequencer was used to created the fastq data This function will allow you to process multiple samples at the same time i e multiple pairs of fastq files For lon Torrent input unmapped bam files are also allowed If you have the Pro Linux version of Target HLA then you can also use sff files as well If using paired reads
97. o 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 you can select multiple fi
98. o single allele candidates others apply to the pair of candidates at a particular locus Single candidate flags e Allele candidates with lower than 90 detection on exons 2 and 3 Class genes or exon 2 Class II genes get an error flag red The detection is the proportion of the exon with sufficient coverage This flag indicates that there may be some key areas of the candidate not well covered by short read data so the result should be treated with caution and may require manual inspection e Allele candidates with lower than 98 detection on exons 2 and 3 Class genes or exon 2 Class Il genes get a warning flag orange e If rare alleles are not ignored option during the analysis then they will be marked with a red exclamation mark in the results e A yellow balance icon next to a homozygous result indicates a possible imbalanced allele in the result This is a warning that there may be an issue with the primer and one of the two candidate alleles is present in a very low proportion of the read data compared to the other i e imbalanced result Using the imbalanced filter these imbalanced candidates can be viewed Paired candidate flags e Where there are multiple allele candidate pairs with very similar coverage and detection statistics these will be marked with a purple pair flag This indicates that while these candidates are not 100 ambiguous i e identical scores they are so close that it is
99. o 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 Jump to a position You can jump to a position in the reference using Jump To Position or jump to one of the annotations within the Target using Jump To Target or Jump to annotation 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 The Jump To Position wizard can be opened by pressing the CTRL and J keys on the keyboard After the wizard is opened you can type in a single coordinate or a pair of coordinates and you can specify a reference contig When a reference contig is not chosen the currently shown contig is used Coordinates can be specified with or without thousand delimiters i e 15200 15 200 and 15 200 are all accepted and mean the same Some examples the currently selected reference contig is chr1 e CTRL J 15200 ENTER gt The Genome Browser display will jump to chr1 15200 e CTRL J 15 200 15 400 ENTER gt The Genome Browser display will show the area chr1 15200 15400 wizard can be opened by pressing the CTRL and J keys on the keyboard After the wizard is opened you can type in a single coordinate or a pair of coordi
100. on Target User Manual Select Start Menu Folder Where should Setup place the program s shortcuts Select the Start Menu folder in which you would like Setup to create the program s shortcuts then dick Next 4 Create a Start Menu folder mixon Target 4 Create shortcuts for all users install4j After clicking the Next button the installation process starts When it is finished the Completing the Omixon Target Setup dialog indicates the success of the installation EE Completing the Omixon Target Setup Wizard Setup has finished installing Omixon Target on your computer The application may be launched by selecting the installed icons Click Finish to exit Setup If you ve decided to create a start menu folder in the previous step Omixon Target shortcut is available now Omixon Target application can be started by clicking on this startup icon or running the executable from application directory Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Peet ures Pictures LD Sticky Notes Music Q Snipping Tool pi Games F Paint Computer A Magnifier s Solitaire Control Panel Devices and Printers amp Mozilla Firefox Default Programs y Omixon Target Help and Support AM Shut down mm Uninstalling the product If you want to remove Omixon Target an uninstaller executable is available in the directory whe
101. on refused error It is acommon configuration problem that the hostname resolves to a different IP on the server e g to 127 0 0 1 via an internal network interface which causes connection deny It is always safe to specify an exact IP address both for the server and clients which is reachable from all the related machines across the network Port Leaving the default port setting is recommended Starting the server Linux Start the server daemon from the command line with one of the following input arguments The available arguments get listed when the server is launched without any Usage omixon target server start stop status restart force reload start stop status restart or force reload Starts server Shuts server down Returns running status Restarts server or if status is stopped if status is running starts if isn t running Automatic startup is not set for Omixon Target Server To set up automatic startup and specify in which runlevel to start the service use a service configuration utility like chkconfig or update rc d Windows To start stop and set startup type for Omixon Target Server launch Control panel Administrative tools Services and modify Omixon Target server service properties By default startup type is set to automatic and the service is run under the System user account MAC OS X Start and stop the server the same way as on Linux Connecting the client Launch client application On Server
102. or rejection This filter will display All variants default then Pending Approved or Rejected variants The visualisation of variants in the short read track can be customized as well Variants are always visible on the nucleotide level but insert and deletion marks can be selectively turned on and off Soft clips can be shown or hidden as well Exporting Genome Browser Data You can Copy to Clipboard the details of individual selected items in the display You can select a read and by clicking on the Copy to clipboard button which can be found on the middle panel in the bottom of your screen you can copy the name and CIGAR string of the selected read to the clipboard Similarly for a selected annotation the position and name of the annotation can be copied to the clipboard You can also copy the reference sequence belonging to a selected item read or annotation by clicking on the Copy reference to clipboard button You can also Capture a Screenshot of the currently visibly tracks within the Genome Browser This will create a png file at your chosen location Working with Tracks Tracks used in the Genome Browser There are two different kinds of tracks used within the Genome Browser Built in tracks can be created using different parts of the Data Analysis module e g importing a reference file or running an alignment or variant call These tracks are not editable and are automatically added to the Omi
103. perating 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 The downloaded installer does not have the permissions to run directly Open a terminal window to make it executable with the following command chmod x omixon target_unix_1_4_0_with_jre sh After that it can be started with the following command omixon target_unix_1_4_0_with_jre sh Installation steps Launch the installer shell script After launching the installer Welcome dialog of the setup wizard appears Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Setup Omixon Target 1 4 0 Welcome to the Omixon Target Setup Wizard This will install Omixon Target on your computer The wizard will lead you step by step through the installation Click Next to continue or Cancel to exit Setup Cancel Please accept the software license agreement to proceed with the installation Setup Omixon Target 1 4 0 License Agreement DY Please read the Following important information before continuing e Please read the Following License Agreement You must accept the terms of this agreement before continuing with the installation SOFTWARE LICENSE AGREEMENT Cuu p gt PLEASE READ THIS SOFTWARE LICENSE AGREEMENT CAREFULLY BEFORE DOWNLOADING OR USING THE S
104. pliSeq Comprehensive Cancer Panel list of target genes csv http tools invitrogen com downloads cms_103573 csv from page http products invitrogen com ivgn product 4477685 1C D search product Here is a description of a run of the kit with some test data if you would like to try it http ioncommunhity lifetechnologies com docs DOC 3043 The amplicons in the kit are documented in a bed file http omixon download s3 amazonaws com AmpliSeqCancerAmplicons 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 AmpliSeq data Analysis Name e g First go Press Next Choose one or more sequencers for your Analysis tip use CTRL or SHIFT click to select
105. re 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 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 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 Best matching candidates i e the best allele candidates based on coverage statistics can be assigned using the Assign Best Matches 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 The results can be exported in TXT
106. re the application was installed If you have created Start Menu items during installation Uninstall Omixon Target icon launches the uninstaller If you intend to use future version of Omixon Target and you don t want your work data to be deleted keep the checkbox unchecked If you wish to cleanup the working directory then selet the option Yes Select Additional Tasks Which additional tasks should be performed Select the additional tasks you would like Setup to perform while uninstalling Omixon Target then dick Next Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Click Next to proceed with the uninstallation process 2 Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Are you sure you want to completely remove Omixon Target and all of its components Click Next to continue or Cancel to exit Setup Finally the installer informs you about the outcome of uninstall Omixon Target 1 4 0 Uninstall Omixon Target Uninstall Omixon Target was successfully removed from your computer Mac OS X installation guide Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 contain Java Runtime Environment JRE e omixon target_macos_1_4 0 dmg If you don t have Java installed on your Mac it will
107. red profiles can be set up automatically by using this function which will also download and import all the data required for each Profile reference sequences known variants and snpEff database 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 security 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 http omixon download s3 amazonaws com target_ref_hg19_chr17 zip CFTR profile e http omixon download s3 amazonaws com target_ref_hg19_chr7 zip The HG19 snpEff database can be downloaded from the following link http omixon download s3 amazonaws com target_snpeff_db_hg19 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 a wrapper around a Target plus a set of reference data and
108. region this will need to be significantly higher at least 2 000 000 Ignore Cross Mappings Some sources of data can be more noisy where there are reads from multiple loci that map to each other Generally the wider the sequencing the noisier the data This option allows reads that have been mapped to multiple HLA loci within the database to be ignored in the results and acts as a kind of noise filter e Ignore Rare Alleles There is now an option to ignore very rare alleles in the analysis they will not be included or reported in this case lf this option is not chosen then these rare alleles will be marked in the results If this option is greyed out you will need to Setup HLA Typing again in order to import the rare alleles database e Typing precision When 8 digit typing is set and both the sequencing and the database have the intronic information then Omixon Target tries to give an 8 digit result Otherwise if falls back to 6 digit e Save read mappings bam format Use this setting to save the mapped reads into a bam format if you would like to browse them in the HLA Genome Browser Sandbox Browser Sandbox Genome Browser Introduction The Sandbox Genome Browser function is for quick visualisation of alignments variants and annotations This module of Omixon Target is available free of charge and can be used as a standalone genome browser The Sandbox Genome Browser was designed with ease of use in mind Multiple alig
109. 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 Warning Flags e Alleles displayed with the blue font are homozygous e Alleles displayed in italics are imbalanced i e present in a much smaller proportion of the reads that the main candidates Results are annotated with warning flags Some of these flags apply to single allele candidates others apply to the pair of candidates at a Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual particular locus Single candidate flags e Allele candidates with lower than 90 detection on exons 2 and 3 Class genes or exon 2 Class II genes get an error flag red The detection is the proportion of the exon with sufficient coverage This flag indicates that there may be some key areas of the candidate not well covered by short read data so the result should be treated with caution and may require manual inspection e Allele candidates with lower than 98 detection on exons 2 and 3 Class genes or exon 2 Class Il genes get a warning flag orange e If rare alleles are not ignored option during the analysis then they will be marked with a red exclamation mark in the results e A yellow balance icon
110. riants and annotations and for simple data analysis tasks e g visual comparison of multiple data sets The Sandbox is a free module The Settings function allows you to manage users licenses and general settings within the tool plus reset the tool to an empty starting state Global Shortcuts The following generic keyboard shortcuts are available in Omixon Target e F1 key opens the Help for the current page or wizard 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 e ALT F4 closes Omixon Target e CTRL C V X copies pastes deletes text in text boxes e SHIFT CTRL left click can be used for multiple selection deselection Data Analysis 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 two pre configured profiles available BRCA e CFTR The pre configured Profile to be used can be chosen w
111. rovided and can be selected in the wizard Custom codon sets can be specified as well in an external file Advanced parameters can be chosen using a text file Call Variants Introduction 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 In the Pro version of OmixonTarget a Samtools mpileup variant call can be selected as well This is the preferred variant calling option for lon Torrent and Roche 454 data Like all wizards in the tool as many options as possible are already preselected within the wizard These are the wizard steps Select Genomic Data Select Sequencer Advanced Options Variant Call Options Variant Effect Options Advanced Options 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 Variant Effect Prediction In the Pro version variant effect prediction can be run within Omixon Target Variant annotations are generated using snpEff SNP Effect Predictor For HG19 chromosomes a variant effect databas
112. rt read alignment with Burrows Wheeler Transform Bioinformatics 25 1754 60 PMID 19451168 e BWA SW Li H and Durbin R 2010 Fast and accurate long read alignment with Burrows Wheeler Transform Bioinformatics Epub PMID 20080505 SnpEff variant annotation and effect prediction tool Cingolani P Platts A Wang le L Coon M Nguyen T Wang L Land SJ Lu X Ruden DM A program for annotating and predicting the effects of single nucleotide polymorphisms SnpEff SNPs in the genome of Drosophila melanogaster strain w1118 iso 2 iso 3 Fly Austin 2012 Apr Jun 6 2 80 92 PMID 22728672 PubMed in process 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 Fast Start e Add Profile Import sequencing fastq data 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 This is the first use fast start tutorial for the Data Analysis module 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
113. rted 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 http www ncbi nlm nih gov SNP 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 Targets Centralised Target configuration for the whole tool
114. s 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 Imbalanced result filtering Using the Setup Filters function you can change the filtering options for the imbalanced allele candidates They are marked when a homozygous result has been assigned and we detect at least one more high quality candidate with a very low proportion of the read data compared to the homozygous allele i e imbalanced result Imbalanced results like these may be due to issues with primers Using the Imbalanced Filter button on the main display these imbalanced candidates can be viewed The minimum imbalanced coverage filter works exactly like the minimum coverage threshold above but only applies to the imbalanced allele candidates The relative imbalanced detection is relative to the best detection score of the main candidates The minimum imbalanced matches controls how many imbalanced allele candidates we display in the table Setting this higher will display more candidates Noise detection filtering Although the primary purpose of the imbalanced allele detection is for the detection of primer issues leading to homozygous results see above the imbalanced candidates can also be viewed for heterozygous results press the Imbalanced Filter again so that all imbalanced candidates are displayed not just the homozygous ones This can be considered a nois
115. s bundled together with a client so there is no need to install a client separately on the server computer To run the server installer system administrator rights are required The server installer can also be run from the command line if no monitor is directly attached please contact support omixon com for details The client only installer doesn t need system administrator rights The steps of the server and client Setup Wizard are equivalent to the desktop version s For both please follow the desktop Installation Guide of your operating system found in the preceding sections of this manual Configuring the server After server installation has finished edit omixon target server vmoptions in Omixon Target application folder to set host and port parameters IMPORTANT The last line in omixon target server vmoptions must be followed by a line feed The default parameters are Domixon server host localhost Domixon server port 4380 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Host Modify localhost value Add hostname e g target server or full domain name e g target server mycompany com or IP address IMPORTANT In the client s connection configuration Server host must be set the exact same value as Domixon server host in the server configuration Make sure that the server hostname resolves to the same IP on the server and client machines otherwise the clients will get a connecti
116. ser Built in tracks can be opened via the Open function found on the side panel These tracks are not editable and are automatically added to the Omixon Target database The other kind of tracks are called User tracks These tracks can be editable and are stored in external non database files Creating tracks User tracks can be created within Omixon Target using the Create Track button in the Manage Tracks wizard or can be imported from external files using the Add Track function in the Manage Tracks wizard User tracks created within Omixon Target are always editable User tracks imported from file can be editable or non editable you can set this when importing the file Annotation editing is currently only supported for files in bed format Modifying tracks Currently track editing is only available for bed formatted editable user defined tracks e To create a new annotation entry use the green button on the middle part of the bottom panel or simply press the Insert button on your keyboard This will automatically generate an annotation entry based on the currently shown browser region You can choose which track you want to add this newly defined entry to from a list containing all the editable tracks and you can also modify the name and end coordinates of the annotation entry by hand e To modify an existing annotation entry by hand you have to select it first this can be done simply by clicking on the entry
117. ser 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 Burrows Wheeler Aligner Homepage of the tool http bio bwa sourceforge net Version used fot testing 0 6 2 Installation with package manager on Debian based systems apt get install bwa Omixon Target Server setup There is a beta version of the Omixon Target Server available for testing Contact sales omixon com if you would like to try this Omixon Target Server accepts multiple client connections simultaneously It allows collaborative work and sharing of analysis results between users It also allows 32 bit clients Windows Linux Mac to be used to connect to a 64 bit server also Windows Linux Mac or Pro server Linux only Configuring the server takes only a few easy steps at first startup Installation Omixon provide two installers for the server version e Server installer containing the server and a single client e Client installer containing a single client only The server i
118. sis parameters Using an existing protocol can speed up the HLA Typing wizard as most of the parameters will be set to those stored in the protocol Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual If you settle on a protocol that works well for your samples you can save this protocol from any successful run and reuse it again later You can also edit existing protocols and create new ones at any time The default protocol cannot be deleted or edited it can be copied Clicking on use for any protocol will launch the main HLA Typing wizard using the parameters from within the selected protocol HLA Typing Protocol Introduction This function is available from the HLA Typing Protocols Dashboard You can view edit and use protocols from here Protocols are stored sets of HLA Typing analysis parameters Using an existing protocol can speed up the HLA Typing wizard as most of the parameters will be set to those stored in the protocol If you settle on a protocol that works well for your samples you can save this protocol from any successful run and reuse it again later You can also edit existing protocols and create new ones at any time Paired Read Options 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 Paired read options For most Illumina data sets the defaults will be fine e Minimum
119. 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 Experiment 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 3 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 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
120. 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 Select the bed or gff file from the file system e Choose the Reference 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 Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 Dashboard Import Annotations G
121. te or area on that contig and jump to the selected location e CTRL S opens the Search sequence wizard Here you can specify a nucleotide sequence to search for in the currently visible reference contig and then step through the hit positions CTRL M enter full screen mode hide all menus and show the browser region in full screen ESCAPE exit full screen mode X switches the browser between horizontal and vertical mode C switches the browser between collapsed and uncollapsed mode T switches the browser between drag and drill navigation mode HLA Manage Tracks In this wizard you can hide and show any tracks The visibility of any track can be set with the Show Track s and Hide Track s functions Hidden tracks are marked with a darker grey color in the Manage Tracks wizard track list HLA Export Results HLA typing results can be exported by clicking on the Export Results button on the HLA Typing dashboard This function generates separate xml formatted files for each sample in the selected Analysis or Analyses These results can be later imported back into Omixon Target using the Import HLA typing results function on the left panel HLA Typing Import Finished HLA typing results can be exported in xml format These result files can later be imported back into Omixon Target using the Import HLA typing results function on the left panel To import the results just check a list of result files and a new Analys
122. 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 are also allowed to process multiple pairs of files for a single sample if you have a very large dataset for a single sample Note you can either process multiple files for one sample or multiple samples but not both at the same time You can chose between copying the selected files into Omixon Target s database by checking the box next to Import data into tool or leaving the selected data in its current disk location this is the default setting 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 result HLA Typing uses 8 digit typing precision Depending on the width of coverage of your data and whether or not candidate alleles have intronic data in the database results can have 6 or 8 digit resolution Protocol Protocols are stored sets of HLA Typing analysis parameters Using an existing protocol can speed up the HLA Typing wizard as most of the parameters will be set to those stored in the protocol If you settle on a protocol that works well for your samples you can save this protocol from any successful run and reuse it again later You can also edit existing protocols and create new ones at any time Paired File Handling Automatic pair handling Paired files can now
123. tion within the Sample Dashboard the goal it to import single sam bam file into the tool The difference with this version is that it will do two things Import sam bam data e Automatically create a new Sample for each imported sam bam file This function will allow you to import multiple sam bam files at the same time Steps in this wizard Select Input File Select Reference Contigs o e e Select a Target Filter this will cause only data that overlaps your target region s to be imported e Select maximum coverage i e choose how much coverage to import The maximum coverage feature should be used with care it will take the first X reads pairs it finds it s not a random sampling method 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 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 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
124. 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 Settings and Functions 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 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 Display setup The variants in the Act Actual variants track can also be filtered This makes use of the Approval features within the tool individual variants can be approved or rejected or still be pending awaiting either approval
125. to the Standard Data Analysis Module for Linux users In addition it also includes integrated access to a number of third party tools including the BWA aligner 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 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 Demultiplexing There are some extra tools for automated demultiplexing of Illumina and Roche 454 data Alignment There is an extra short read aligner BWA Variant Calling There is a another variant calling pipeline based on SAMtools mpileup which gives better results for lon Torrent or Roche 454 data HLA Typing Module Optional Omixon Target contains simple fully automated HLA Typing algorithms which have been developed to work for reads produced by all three major sequencing platforms It is possible to do HLA typing from any kind of experiment including targeted sequencing whole exome or whole genome experiments The HLA Typing Module works on Windows 64 bit recommended Linux 64 bit recommended and Mac OS X This Module contains only the HLA Typing feature of Omixon 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 s
126. ts Approve Variants Approve Sample 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 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 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 Introduction If you have imported sequencing data then you can map and align this data against a Reference Genome using this function This version of the Map and Align runs with a single sample This function has four analyses built in to it Map and Align using Omixon mappers Map and Align using Burrows Wheeler aligner only in Target Pro edition Variant Call using GATK optional preferred for Illumina data Variant Call using Samtools only in Target Pro edition preferred for lon Torrenet and Roche 464 data Variant Effect Prediction using SnpEff
127. ull screen mode hide all menus and show the browser region in full screen ESCAPE exit full screen mode X switches the browser between horizontal and vertical mode C switches the browser between collapsed and uncollapsed mode T switches the browser between drag and drill navigation mode INSERT insert new annotation ENTER edit selected annotation DELETE delete selected annotation M move selected annotation to start at the locked cursor position R resize selected annotation to end at the locked cursor position Sandbox Open File In this wizard you can visualise multiple alignments and create an annotation and variant track in a single step Reference files must be in fasta or multifasta format Note that a built in HG19 reference is available within the Sandbox Genome Browser so if your alignments annotations or variant calls are HG19 based no reference file is need to be imported you simply have to select a list of alignment and or annotation and or variant call files and open them all in one step Annotations must be in gff or bed format Annotation files selected in this wizard are merged and visualised in a single track For visualising multiple tracks separately use the Track Manager function Alignment files must be in sam or bam format unsorted and or unindexed files are also accepted and are automatically sorted and or indexed during the import Tracks imported in this wizard are called built
128. ware requirements for all the optional modules are the following 64 bit multi core CPU with 64 bit operating system atleast 8GB memory Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual 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 The following sections contain the descriptions for the installation steps required for each operating system The Omixon Target Pro version of the product has a separate section to summarize installation and setup instructions related to this specific type of distribution After installation confiuration steps for the server and client application of Omixon Target Server are also described separately Windows installation guide We provide two versions of the installer package for Windows operating systems both are bundled with a Java Runtime Environment JRE e 64 bit version named
129. xon Target database The other kind of tracks are called User tracks These tracks can be editable and are stored in external non database files Creating tracks User tracks can be created within Omixon Target using the Create Track button in the Manage Tracks wizard or can be imported from external files using the Add Track function in the Manage Tracks wizard User tracks created within Omixon Target are always editable User tracks imported from file can be editable or non editable you can set this when importing the file Annotation editing is currently only supported for files in bed format Copyright 2013 Omixon Biocomputing Kft Omixon Target User Manual Modifying tracks Currently track editing is only available for bed formatted editable user defined tracks e To create a new annotation entry use the green button on the middle part of the bottom panel or simply press the Insert button on your keyboard This will automatically generate an annotation entry based on the currently shown browser region You can choose which track you want to add this newly defined entry to from a list containing all the editable tracks and you can also modify the name and end coordinates of the annotation entry by hand e To modify an existing annotation entry by hand you have to select it first this can be done simply by clicking on the entry with the left mouse button The selected annotation element is marked by a li

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