Alamut Genova User Manual (EN) Version 1.4 USER MANUAL

Transcription

1 Alamut Genova User Manual (EN) Version 1.4 USER MANUAL Last modified date: 3rd of December 2018

2 2 Table of Contents Preamble 6 Contact Us Enhanced Features 3. Introduction Prerequisites Operating System Internet Connection Hardware Requirements License Type Installation Step-by-Step Configuration License Network View Laboratory Variant Databases Classification Misc Patient Report Homepage Overview Interface Interface: Shortcut Buttons Interface: Menu Options Applications File View Web Variant Tools Help Reported Problems and Solutions Genomic View Chromosome View Overview of Chromosome Overview of Transcript Select a Transcript... 37

3 3 Table of Contents 6. Direct Gene Search Variant View Main Variant Window and Variant Display Navigation Creating a Variant Variant Properties Variant Panel Information Tab Annotation Tab Splicing Tab Splicing Tab Display Splicing Tool Menu ESP Prediction Tab Options Highlight Differences Report Splicing Track Background on Prediction Methods Set of Human Constitutive Exon/Intron Junctions References Report Tab Import and Create a Variant Database Import Create Manage Variant Datasets Sample Pages Protein Structure View View from Transcript View from Variant D Protein Structure Window Track Configuration and Settings Application Settings Track Configuration Creating a New Track Configuration Deleting a Track Configuration Switch Between Track Configurations... 69

4 4 Table of Contents 10.3 Modifying Tracks Add Track(s) Selecting Information Ordering Track(s) Deleting Track(s) Tracks Allele Frequency Databases Classified Variant Databases Display Genes Nucleotide Position Transcript Options Create Variant and Create Sequence Annotation Genome Laboratory Variant Databases Nucleotide Conservation Orthologues Sequence Annotations Protein Domains Protein Secondary Structure Protein Variant Regulatory Regions Repeat Masker Somatic Structural Variants Splicing Predictions Sanger Electropherogram Track BAM Alignments Loading BAM Alignments BAM Track Display and Tools BAM Track Settings Base and Read Display Settings Load BAM and VCF Files Preferences BAM Preferences Private Sequence Annotation 91

5 5 Table of Contents 12.1 Annotate Nucleotide and Nucleotide Sequence Data Source Data Source List External Variant Databases External Variant Databases: Track Display External Variant Databases: Access References Programatic Access Programmatic Access For Software Providers Index 0

6 Preamble

7 7 Preamble 1 Preamble PLEASE READ CAREFULLY Alamut Genova is a component of the Alamut Software Suite, a set of applications dedicated to genomic variant annotation, filtration, and exploration. Alamut Genova does not provide recommendations for medical diagnosis. It must be used by human genetics professionals and with critical judgment. Interactive Biosoftware cannot guarantee the accuracy of information and predictions it provides. 1.1 Contact Us To get support for Alamut Genova, you can contact us: 1. You can send an to Technical Support team via the homepage of Alamut Genova: Click Help on the Alamut Genova homepage menu, click Contact Support (support@sophiagenetics.com 7 ). 2. Or you can contact support directly using the following address: support@sophiagenetics.com.

8 Enhanced Features

9 9 Enhanced Features 2 Enhanced Features New Features Genomic View 34. CNV in Structural Variants /Database of Genomic Variants (DGV): The Copy Number Variation (CNV) appears in the Database of Genomic Variants (DGV) track. 84 Functions 3D Protein Structure 63. Sanger Electropherogram Track 86. We have improved the functionality and visual display of variant annotation by creating the Variant Panel 45. On the Annotation Tab we have added: (Variant View 42 ; Create Variant Panel 45 ; Annotation 46 ). o The Human Phenotype Ontology (HPO). o Suggested ACMG/AMP Classification 22. Predictions Track 84 You can now see splicing predictions on a track. 44 ; Variant

10 Introduction

11 11 Introduction 3 Introduction 3.1 Prerequisites The installation of Alamut Genova needs the following technical specifications Operating System The program is available for the following operating systems: Microsoft Windows 10, 8, 7 and 32 and 64-bit versions. The program is available as an Installer program (.exe) or self-extractable archive (.exe) or compressed file (.zip). Mac OS X Snow Leopard/Lion/Mountain Lion/Mavericks/Yosemite/El Capitan (Mac OS X ). Note: For Windows 32 bit and Mac OS users not all features are available Internet Connection An internet connection is required to connect to the Interactive Biosoftware s database server: Connection to the following IP addresses are required: ; ; ; ; The software handles connections through HTTP on port 80 and 443, optionally through a proxy Hardware Requirements Computer: Intel Pentium III or later processors 1 GB RAM 30 MB free disk space. Display screen resolution: 800 x 600 pixels. The software does not alter system directories or the registry. Write permissions are required on the software directory to ensure continued functioning of the application and user parameters.

12 12 Introduction 3.2 License Type Alamut Genova is installed with a floating license. Floating licenses can be installed on multiple computers, with a limited number of concurrent users and are managed via an extranet. 3.3 Installation Step-by-Step Alamut Genova extranet page: Once an Administrator login has been created, the IT Administrator must log-on to the Alamut Genova extranet page and create New User Accounts. The user will receive the Institution ID, User Name and Password via . In order to install the software the user must now open the Alamut Genova extranet page and complete the Sign-in with the Institution ID, User Name and Password provided.

13 13 Introduction Begin Installation o Click Downloads, click Installer. Follow the installation instructions in accordance with your computer operating system: o Windows 10, 8, 7 and XP (known to work also on Windows Vista), 32 and 64-bit versions. Download the Alamut Genova Installer or Self-Extractable executable (.exe) from our extranet page: Installation. If you have downloaded the Installer: execute it and choose an installation folder where you have write permissions.

14 14 Introduction If you have downloaded the Self-Extractable executable (.exe) file, simply double-click the installer to launch the installation program and follow the instructions presented to you. Select a folder where you have write permissions. Post-Installation. After you have finished installing the program you should remove the files used for the installation: the Installer file or the Self-Extractable executable (.exe) file. o Mac OS X Snow Leopard/Lion/Mountain Lion/Mavericks/Yosemite/El Capitan (Mac OS X ) Download the Alamut Genova(.dmg) file from our extranet page: Alamut Genova for Mac is available as a disk image. Note: For Mountain Lion (OS X 10.8), Mavericks (OS X 10.9), Yosemite (OS X 10.10) and El Capitan (OS X 10.11) users: this External Web Page 11 describes how to enable installation of applications from sources other than the Mac App Store. Un-package. Before any software can be installed, it must first be un-packaged. A disk image can be thought of as the virtual equivalent of a CD. The Alamut Genova application in the disk image is contained within a single file: for instance "AlamutGenova dmg" You then "insert" or "mount" the disk image into the machine by double-clicking the file. Having done this, the disk image will appear as another device in the Finder. Installation. The final step of the process is to actually install the software where you have read-write permission. An application bundle to install Alamut Genova is provided. All you have to do is copy the program to your desired location (usually your Applications folder) and run it. Copying the program is performed simply by using drag and drop. Post Installation. After you have finished installing the program you should remove the files used for the installation. You should unmount the disk image that was used in the installation process. This can be done by using the eject icon next to it in the Finder sidebar. You can also drag the mounted disk icon to the Trash. Set up Alamut Genova with your credentials: o Inside the folder where Alamut Genova has been installed, find the AlamutGenova file, and double-click on it to launch the program. Note: For Linux users, double-click on the file AlamutGenova.sh to launch the program. o Accept the End User License Agreement.

15 15 Introduction o The Application Settings window will open. Please ensure that you have completed the information required on each of the following tabs in order to ensure the software runs effectively: License, Network, if applicable Misc (HGMD Credentials). See Configuration Configuration When first launching, the Application Settings window will open automatically. Note: Thereafter you can access the Application Settings window by clicking on the Application Settings icon on the toolbar on the main page. The Application Settings window includes the following tabs: License 16 Network 19 View 20 Laboratory Variant Databases 21 Classification 22 Miscellaneous 23 Patient 24 Report 25

16 16 Introduction License User: On the first launch of Alamut Genova the following must be completed in the user section of the License tab: o Institution. o License key. o User Name. o Password. (Click Apply before leaving the tab) These details will be saved and completed automatically in future log-ins. Language: Select your preferred language (English or Français). Application: Tick the box to automatically check for updates or un-tick to update manually. o To manually download updates you can either:

17 17 Introduction Connect to our extranet page ( 16 ), select the Downloads section and if available download the updated version shown. Or on your computer find the Alamut Genova Installation Directory and click the Updater.exe file. The Maintain Alamut Genova Installer will launch, here select Update Components and follow the instructions. Each time the user logs-in they will have to enter their Username and Password. The log-in window also includes a warning for users "This software application is a genomic variant exploration system that does not provide recommendations for medical diagnostics. It must be used by human genetics professionals and with critical judgment."

18 18 Introduction When a user logs-in to Alamut Genova for the first time they have to agree, by clicking "I agree" to the Alamut Genova License Agreement.

19 19 Introduction Network To connect via proxy complete the necessary information in the Network tab (this may require the input of your IT administrator). o Local Server Port: by default Alamut Genova will select the server port, however if you have another application using the same port, Alamut Genova will not work, here you can modify the port in order to run the software. o Use Proxy: Tick the Use Proxy box to complete the necessary proxy details.

20 20 Introduction View The View tab in the Application Settings window is where you can modify the track configuration and select the information you wish to include within the tracks. See Track Configuration & Settings 67. o Show Selected Transcript on Variant Tracks: select/deselect to view the selected transcript on the tracks. o Description of Stop Codons: Select from X or HGVS 2.0 standard nomenclature Default Genome Build. o GRCh37 or GRCh38: Select which genome build you would like to use. View. o The view section is where you can modify the information displayed on the tracks. o Track Configuration: Select which Track Configuration 67 you wish to modify. See Track Configuration to view details on how to modify tracks and their configuration.

21 21 Introduction Laboratory Variant Databases See Import and Create Variant Database. o When saving a variant the data is stored in the Laboratory Database by default at a default location on your computer. This can be set by clicking on the Plus button to create a database. o Within the database there are two types of datasets: Variant Datasets and Sequence Annotation Datasets. Variant Datasets contain single variants created by the user and Sequence Annotations datasets contains variant sequences and regions created by the user. o When creating a personal database make sure it saves as a.db file. o If you change operating system; save all your created databases on the installation repertoire this way they can be moved with the Alamut Genova files. 59

22 22 Introduction Classification Pathogenicity Classes Scheme (ACMG/AMP like). o Personalize the names of the pathogenic classifications.

23 23 Introduction Misc Mouse Wheel: two mouse scroll options are available. Sanger 86 : for your Sanger Sequencing data you can set your required Quality Threshold.

24 24 Introduction Patient On the Patient Tab you can select which information to include on a Patient Report.

25 25 Introduction Report In the Report Tab you can select what information you wish to include by default in a variant report 58.

26 26 Introduction 3.5 Homepage Overview On the homepage there there are a number of Menu Options 27, a bar of Shortcut Buttons 26 and the Go to search bar. On the left-hand side of the homepage a number buttons to improve the user experience of including: Open Gene; Open Genome View; Open Mitochondrial View; Video Tutorials; User Manual; Quick Start; About Alamut. The information box on the bottom left of the page contains information crucial for the user such as: the version of Alamut Genova; last update date; license expiry date (Valid until); and the number of available genes. The following Interface 26 section will describe each of these functions in more detail. 3.6 Interface In this section we will cover the interface display of Alamut Genova, how to navigate the Main Window and the options that are available from the Menu & Shortcut Buttons Interface: Shortcut Buttons These are the available buttons displayed on the Interface of Alamut Genova. o Settings and Quick Access Buttons. & Select which human genome build you wish to view.

27 27 Introduction Opens the mitochondrial genome for your default genome build. Opens the Gene Selection window, where you can choose a gene to study. Opens the Home Tab. Opens the Application Settings window. Selects forward or reverse to change the reading direction of the selected strand. Opens the Genetic Code window which displays the Standard Genetic Code and the Mitochondrial Genetic Code. Opens the Amino Acid Comparison window. o Web Shortcuts. The web shortcuts on the toolbar are also available in the Weblist box on the Menu section. Displays the selected genomic region in the Ensembl Browser Displays the selected genomic region in the NCBI Sequence Viewer website. Displays the selected genomic region in the UCSC Browser Displays the HGNC Symbol Report for the selected gene. Displays the selected gene on the OMIM website. Displays the selected gene on the GENATLAS website. View the selected gene in the Gene Reviews website. View the UniProt entry for the product of the selected gene. o Go to: Menu Search an official gene symbol. The Transcript Selection window will open where you can select the transcript you wish to study, variant data, sequence data will be loaded on the tracks Interface: Menu Options Application, File, View, Web, Variant, Tools and Help.

28 28 Introduction Applications You can find: Open Gene: to load the Gene Selection window (to select a gene to study). GRh37 : to display genomic data in GRCh37. GRh38: to display genomic data in GRCh38. Mitochondrial Genome: to display mitochondrial genomic data. Home: to return to the Homepage. Settings: to open Application Settings window (which includes: License, Network, View...etc). Exit: to exit & close Alamut Genova File Import Almaut Visual mutation files...: to import variant (*.mut) files from Alamut Visual into Alamut Genova. Import VCF File: to import a Variant File into your dataset (*.vcf) in Alamut Genova. Import BED File: to import a BED Region File (*.bed) into Alamut Genova. Import GFF File: to import a GFF Region File (*.gff) into Alamut Genova. Open VCF File: to import a Variant File (*.vcf*). Open BAM File: to import a BAM File (*.bam). Open Sanger File: to import a Sanger File (*.ab1). View File Content: to view all available variants. Export Fasta Sequence: to export a genomic sequence, cdna Sequence or a Peptide sequence.

29 29 Introduction View Toolbars : to open the submenu that allows you to select/deselect that toolbars you wish to view on the homepage. Focus on gene: When two or more genes are displayed on the Genes track you can click on this tool to view a list of genes available (zoom out to include more genes). Focus on...: to open the Focus On window, which allows you to search for a specific Region, Genomic Coordinate, cdna Coordinate, Protein Coordinate, Variation, Nucleic Sequence and Protein Sequence Note: The search will only be undertaken on the genomic data of the currently displayed gene/genomic region. Forward direction & Reverse direction: Select to change the reading direction of a gene on the track display. Show Ruler: to display or remove the ruler tool on the main window. The ruler displays genomic positions and includes a marker tool that can define the distance between two selected genomic positions. Click on the first position then click on the marker button (the A marker will appear on the selected position), select the second position (the B marker will appear on the selected position).the distance between the two positions will be displayed on the ruler. You can click and drag the A & B markers to select different positions.

30 30 Introduction Color Nucleotides: Select/Unselect to display nucleotides in color or in black. Use Amino Acid 3 Letter Code: Select/ Unselect to display 3 Letter Code of Amino Acids. Full Screen: Click to view Alamut Genova in full screen. Increase Font, Decrease Font and Reset Font: Select to modify font size on Alamut Genova Web Select from the list to view a studied sequence or gene in an external database or viewer Variant New Variant : a nucleotide/nucleic sequence, click New Variant to create a variant from your chosen sequence. View All Variants: to view the Samples window, which displays all databases & datasets, all variants within your available databases.

31 31 Introduction Tools Genetic Code: to launch our Genetic Code window which displays the Standard Genetic Code and the Mitochondrial Genetic Code. Compare Amino Acids: to view amino acid structures and compare properties. Assembly Mapping: Maps data to a specific genome build or assembly Help Software Documentation. Data Sources. License Agreement. Software References: how to cite Alamut Genova in a publication. Contact Support: to send an to our Customer Support team an (support@sophiagenetics.com). About Alamut Genova: to see a brief explanation of Alamut s name. 3.7 Reported Problems and Solutions Q: Alamut Genova does not save my options or prompts the Application Settings window when launched.

32 32 Introduction o A: You must have the rights to write to the Alamut Genova folder, check with your IT Administrator. Q: Alamut Genova can t reach the network or a network error occurs. o A: This issue may have many causes due to your local configuration that must be checked by your IT Administrator. o Examples include: Wrong proxy settings. Firewall blocking the Alamut Genova internet address. Anti-virus preventing Alamut Genova from accessing the internet. Data loss prevention software blocking the sending of unencrypted pid. Recent changes to your computer or networking system. Q: Alamut Genova installer or Alamut Genova program does not operate. o A: This issue may occur due to your local security policy (e.g. sandbox) and must be handled by your IT Administrator. Q: Alamut Genova cannot load certain genes. o A: Ask your IT Administrator to check that the proxy size limit is high enough and that proxy cache has been disabled.

33 Genomic View

34 34 Genomic View 4 Genomic View The Genomic View allows you to visualize all chromosomes including the mitochondria, intergenic genomic regions, structural variants and extragenic regulatory regions. The Genome build you view is based on your default preferences in the settings either GRCh37 or GRCh38. The entire genome can be viewed, with a list of its corresponding chromosomes. From the Genomic View you can select the chromosome you wish to study, by double clicking on it. Clicking on the Mitochondrial Genome opens a new tab, with a newly designed overview for the genes of the circular Mitochondrial Genome.

35 Chromosome View

36 36 Chromosome View 5 Chromosome View In this section we will delve into the different viewing options available within the chromosome view: Overview of Chromosome 36 and Overview of Transcript Overview of Chromosome When you have selected a chromosome from the Genomic View 34 the top section of the window will display an overview of your selected chromosome. On the Overview of Chromosome, click a cytogenetic band on the chromosome in order to load the relevant data. Zoom in on the Genes track using your mouse scroll-wheel in order to view the genes available in the selected band. The genes are displayed with named tags along the strand. Note: The reading direction of the gene (i.e. the DNA strand) can be changed by using the forward direction 5.2 and reverse direction icons on the toolbar, in order to make it easier to study it. Overview of Transcript Click on a selected gene to study. The transcript of your selected gene is displayed in the overview, with the exons in blue and introns in yellow.

37 37 Chromosome View Click on an exon to load the coding region you wish to study. 5.3 Select a Transcript On the Genes track 76, zoom in and select a gene then right-click to view the transcript submenu options. You can select to view your Preferred Transcript. When clicking View All, all available transcripts will appear on the track directly underneath the default transcript. To change the Exon Naming, when you have selected your preferred transcript click on "Set Exon Naming", which will appear under "Set working transcript", if both Systematic and Custom naming is available for this transcript.

38 Direct Gene Search

39 39 Direct Gene Search 6 Direct Gene Search The fastest way to access a transcript is through the Gene button Click the Gene button on the toolbar. to launch the Gene Selection window. On the Gene Selection Window: o You can type the name of your gene of interest in the search bar or select a gene from the shortcut list. o You can also edit the genes displayed in the shortcut list, or create a new gene list, by ticking the "change shortcut" box. o Gene Request: If a gene or transcript version is not available, send our team an request. o The Transcript Selection widow will open. Alamut Genova will highlight the canonical transcript by default in the transcript list.

40 40 Direct Gene Search Tick the "Preferred Transcript" box to select your preferred transcript.

41 Variant View

42 42 Variant View 7 Variant View With Alamut Genova you can annotate existing variants and create new variants. In this section we will cover the necessary steps to import & manage your variant datasets, to create and annotate variants, in addition to generating a report using the Variant Panel Main Variant Window and Variant Display A new tab will open each time you open a transcript or create a variant. The tab will be names after the transcript or variant created e.g. "MLH1 NM_..." You can open several tabs at the same time Navigation Once you have selected your transcript of interest, to ease page and track navigation, use the following: o Zoom in: Use the scroll wheel on your mouse to zoom in/out (in order to view the variants clearly on the tracks). o Move up/down: Click and Drag up/down, or press and hold the CTRL button and use the mouse scroll wheel. (There is also a scrollbar at the right-hand side of the window). o Move left/right (along the gene track): click and drag left/right. o Zoom in/out by using the mouse scroll-wheel. Using the arrow keys: o Move left/right (along the tracks): Use the left or right arrow key to move along the tracks. o Zoom in/out: Use the up arrow key to zoom in and the down arrow key to zoom out.

43 Creating a Variant

44 44 Creating a Variant 8 Creating a Variant Alamut Genova allows you to create a variant and add it into the database with annotations you would like to include. o On the Genome and Gene track: Click to select nucleotide(s). Right-Click on your selection, click to select Make Variant. Other options on the submenu: o Make Sequence Annotation: Select to annotate a nucleic sequence (See Private Sequence Annotation 92 ). o Copy: This function copies the highlighted nucleotide(s) to enable the user to paste the sequence elsewhere. o Copy rev/comp: This tool will copy the selected nucleotide(s) and when you paste, the original sequence will appear followed by the reverse-complementary counterpart. o Copy genomic position: Select to copy the genomic position of your selected nucleotide(s). o Copy cdna position: Select to copy the coordinate position along the track of your selected nucleotide(s). o Copy Protein position: Select to copy the protein position of your selected nucleotide(s). 8.1 Variant Properties Variant creation and annotation begins with the launch of the Variant Properties window. On the window select the variant type: Substitution, Deletion, Insertion, Duplication, Delins and complete the necessary data or coordinates, click OK.

45 45 Creating a Variant 8.2 Variant Panel Once a variant is created Alamut Genova launches the Variant Panel, which contains the tabs: Information, Annotation, Splicing and Report. Note: You can navigate between tabs, even whilst adding information, by clicking on each tab Information Tab In the Information Tab you have access to the information available on your selected variant: Genomic Level, Protein Level, Transcript Level, External Tools, Public Databases and Missense Predictions.

46 46 Creating a Variant Annotation Tab Open the Annotation tab. Note: To open the Annotation tab: Select your desired nucleotide(s) + right-click on your selection, click Make Variant, complete necessary data on Variant Properties + click OK, select Annotation tab.

47 47 Creating a Variant The Annotation tab contains: o Database & Sample: Select where you wish to save your newly created or annotated variant. o Comment: Enter in free-text form any comments about the variant you wish to include. o Export: Click to export the results of the Annotation Tab, Information Tab, Splicing Tab and Report Tab to a Word or Excel file. Select the template file of your choice and select the file you would like to export. o RNA Analysis: Enter in free-text form RNA analysis notes. o Other Occurrences: Shows if variant exists in other databases or samples. o Patient Phenotype: Enter in free-text form any Patient Phenotype information you wish to include. In order to make the variant annotation process easier the Alamut Genova Annotation Tab now includes additional functionality. o Variant History: This section shows a list of previously made annotations on the specific variant you have selected to work on. This enables you to see an edited history of the variant, particularly helpful when more than one user is using the software. o Classification: Select the pathogenic classification of the variant and as a new feature, access the ACMG/AMP Standards and Guidelines tool to enable the user to classify the variant. Click on Show Details to access the ACMG/AMP Standards and Guidelines window. The AGMG/AMP pathogenicity classification of a variant needs to be carried out by human genetics professionals and with critical judgment, using additional information about the patient, their phenotype or environment, it is not possible to make a definitive conclusion without verifying the results experimentally with approved genetics tests.

48 48 Creating a Variant HPO Phenotype: The Human Phenotype Ontology (HPO) allows you to select from a list of phenotypes, enabling the user to include in the annotation. Click Edit: in the Human Phenotype Ontology window select your desired phenotypes from the list or search in the search bar. Click OK.

49 49 Creating a Variant You can export your variant data in either word or Excel format, using the export button at the bottom of the annotation tab. You will be asked to select an Excel or Word template file and a destination file. Alamut Genova will read the template file, populate cells named with an annotation name, and will output the populated spreadsheet or word document in the destination file Splicing Tab Alamut Genova includes a splicing module integrating a number of prediction algorithms and splicing prediction data: Splicing Signals: o MaxEntScan Donor, acceptor o GeneSplicer- Donor, acceptor o NNSPLICE- Donor, acceptor o Known Constitutive Signals- Donor, acceptor o Splice Site Finder-like (SSF) Donor, acceptor, branchpoint

50 50 Creating a Variant o Mercer et al. high-confidence branchpoints o Limnos- branchpoints Exonic Splicing Enhancers (ESE) binding site detection: o ESEFinder o RESCUE-ESE o EX-SKIP The Splicing Tab displays reference (wild-type) and mutated sequences, and the splicing predictions Splicing Tab Display Exons are drawn as grey boxes. Hits from SpliceSiteFinder-like, MaxEntScan, NNSPLICE and GeneSplicer are displayed as blue vertical bars for 5 (donor) sites, and as green vertical bars for 3 (acceptor)sites. The height of each bar is proportional to the maximum possible score computed by the corresponding algorithm. Known constitutive signals are displayed as small blue 5 or green 3 triangles, close to the sequence letters. When moving the mouse over each vertical bar or triangle, a tooltip appears with the corresponding score. You can display score numbers for each hit bar by clicking the bar itself.

51 51 Creating a Variant A Splicing Track has additionally been added to Alamut Genova. Splicing Tool Menu The Splicing Tab includes a Splicing Tool Menu with the following tool buttons: o ESE Predictions 51. o Options 52. o Highlight Differences o Report 53. o Copy Snapshot ESP Prediction Tab To display the ESE Predictions Tab. o The ESE hits from ESEFinder are displayed above each sequence, and RESCUEESE hexamers are shown below the sequence.

52 52 Creating a Variant o The ESE Predictions Tab includes its own tool menu. o ESE Predictions: Click / Unclick to switch between viewing the ESE Predictions Tab and Splicing Tab. o EX-SKIP: Click to launch EX-SKIP tool. o Options: Click to launch the Splicing Predictions Options window. o Highlight Differences: Click to reveal differences between wild-type and mutated scores. o Report: Click to generate a separate report of the ESE Predictions. o Copy Snapshot: Click to take a snapshot of the ESE Predictions Tab. EX SKIP TOOL INFO: The pre-filled web form of the EX-SKIP tool is displayed in a new window. Input sequences are created as follows by Alamut Genova: only exonic sequences are taken into account, with up to 30 exonic nucleotides before or after the variant position within the exon Options To select which predictions to display and to modify thresholds use the Splicing Predictions Options window. Click the Options button in the Splicing Tool Button Menu. To save changes to the splicing options click apply.

53 53 Creating a Variant Highlight Differences To reveal differences between wild-type and mutated scores. Click the Highlight Differences button on the Splicing Tool Button Menu. Unchanged scores fade, while scores are displayed beside those that differ Report To generate a splicing prediction report. Click the Report button on the Splicing Tool Button Menu, complete the Flanking Region info. The report is generated in HTML web format. It can be later opened and edited by most word processors.

54 54 Creating a Variant Splicing Track The Splicing Track allows you to view the splicing predictions whilst on the Chromosome View or Gene View. To add the Splicing Track. o Click the Application Settings icon, click View, click Add Track (button). o In the Select Track window. Select Splicing Predictions, and click OK. When returning to the Application Settings window, click Apply, click OK. To view the display of the Splicing Track see: Splicing Predictions Background on Prediction Methods SpliceSiteFinder-like is a method based on position weight matrices computed from a set of human constitutive exon/intron junctions for donor (both GT and GC) and acceptor sites. Alamut Genova uses the matrix described by Zhang et al. (1998) for branch points and the algorithms described in Shapiro et al. (1987). MaxEntScan is a method based on the Maximum Entropy principle, developed by the Burge Lab at MIT and described in Yeo et al. (2004). The MaxEntScan splice site datasets and algorithms are fully integrated inside Alamut Genova, with permission

55 55 Creating a Variant from Christopher Burge. Alamut Genova only reports scores from the Maximum Entropy Model. NNSPLICE (available at the Berkeley Drosophila Genome Project website is a prediction method based on neutral networks (Reese et al. 1997). Although not fully integrated inside Alamut Genova, it is transparently queried from within the software. Alamut Genova reports scored from NNSPLICE 0.9. GeneSplicer, an Open Source software available from the University of Maryland CBCB, combines several splice site techniques; among them Markov models (Pertea et al.2001). Known Constitutive Signals Alamut Genova reports in the splicing module each occurrence of the 9-mers (3 exonic + 6 intronic nucleotides) found in the donor subset of human constitutive exon/itron junctions, and each occurrence of the 6mers (4 intronic + 2 exonic) found in the acceptor subset. Mercer et al. High confidence branchpoints uses exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and by split and inverted alignment, Mercer et al. (2015) identified 59,359 high-confidence human branchpoints in >10,000 genes, thus providing a first map of splicing branchpoints in the human genome. Limnos: Alamut Genova has added a new splicing predictor called Limnos. Limnos predicts branchpoints by using logistic regression. ESEFinder uses a method that computes putative binding sites for Exonic Splicing Enhancers (Cartegeni et al. 2003). We have embedded these ESEFinder matrices (licenced from Cold Spring Harbor Laboratory) inside Alamut Genova so as to perform the same computation as that provides by the CSHL ESEFinder website In the RESCUE-ESE approach, specific hexanucleotide sequences are identified as candidate ESEs (Fairbrother et al., 2002). The set of human hexamers available from the RESCUE-ESE website are embedded inside Alamut Genova. EX-SKIP compares the ESE/ESS profile of a wild-type and a mutated allele to quickly determine, which exonic variant has the highest chance to skip this exon. It calculates the total number of ESSs, ESEs and their ratio. It computes specifically the number of RESCUE-ESEs (Fairbrother et al. 2004; Fairbrother et al.,2002) FAS-ESSs (Wang et al., 2004), PESEs/PESSs (Zhang et al., 2004), neighbourhood inference (Stadler et al. 2006) and EIE/IIEs (Zhang et al., 2008) for each segment. The EX-SKIP tool is available through Alamut Genova s ESE Predictions Tab.

56 56 Creating a Variant Set of Human Constitutive Exon/Intron Junctions We have gathered a set of human constitutive exon/intron junction sequences as follows: From the RefSeq database (as of Dec. 2007) and with a reviewed status, 10,728 human mrna sequences were mapped onto the human reference genome (NCBI 36). Based on this mapping, genomic exon/intron boundary sequences were extracted into separate subsets for donor and acceptor sites. With these sequences, we have built three position weight matrices: two matrices for donor sites (GT and GC sites), and one matrix for acceptor sites (AG sites)

57 57 Creating a Variant References Users may also refer to a book chapter presenting in silico splice tools integrated in Alamut Genova: In silico prediction of splice-affecting nucleotide variants in In Silico Tools for Gene Discovery, Springer, Cartegni et al. ESEfinder: A web resource to identify exonic splicing enhancers 57. Nucleic Acids Res (2003) vol. 31 (13) pp Fairbrother et al. Predictive identification of exonic splicing enhancers in human genes 57. Science (2002) vol. 297 (5583) pp Hellen Splice Site Tools: A Comparative Analysis Report 57. NGRL Manchester Report 2009 Houdayer et al. Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants Hum Mutat Aug;33(8): Mercer at al. Genome-wide discovery of human splicing branchpoints Genome Res (2015) 25(2): Pertea et al. GeneSplicer: a new computational method for splice site prediction 57. Nucleic Acids Res (2001) vol. 29 (5) pp

58 58 Creating a Variant Raponi, M., Kralovicova, J., Copson, E., et al. Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon Hum Mutat. (2011), 32, Reese et al. Improved Splice Site Detection in Genie 57. J Comp Biol (1997) vol. 4 (3), pp Shapiro, M. B. and P. Senapathy (1987). RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15(17): Yeo et al. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals 57. J Comput Biol (2004) vol. 11 (2-3) pp Zhang et al. Statistical features of human exons and their flanking regions 57. Hum Mol Genet (1998) vol. 7 (5) pp Report Tab In the Report Tab you can view and select, on the left-hand side of the screen, the information you wish to include in your report. On the left-hand side of the page you can select the information you wish to include by clicking /unclicking on the button with the topic name. o The topic buttons include: o HGVS. o ACMG/AMP Guidelines. o Additional Information. o External Variants. o Splicing. o Splicing Screenshot. o Protein Information. o Annotations. o Occurrences. o Orthologues. If you wish you can generate and save the variant. By clicking the right mouse button you can save and print your report.

59 59 Creating a Variant 8.3 Import and Create a Variant Database Alamut Genova allows you to import and create a database with a collection of variant datasets and/or sequence annotation datasets. You can create tracks that display the information you have created or imported Import To import a database from your device into Alamut Genova. o Open the Application Settings 67 window. o Select the Laboratory Databases 21 tab, click on the plus button, select the database from your files, select the option of Variant dataset, (If the database includes Variant and Sequence Annotation datasets select both). Note: The imported file must be formatted as a VCF file.

60 60 Creating a Variant Alamut Genova will take you back to the Application Settings page click Apply, click OK. Your imported database will be added to the Laboratory Variant Databases 21 track, you can add the database to the tracks. o Within the database there are two types of datasets: Variant Datasets and Seqeunce Annotation Datasets. Variant Datasets contain single variants created by the user and Sequence Annotations datasets contains variant seqeucnes and regions created by the user. Create To create a personal database open the Application Settings 67 window. On the Laboratory Databases tab, click Create Database (button), select a directory folder, name the new database, click Save. Note: make sure the file type is Database (*.db). The Create Database pop-up will appear, click Create. Select your newly created database, on the Dataset section select whether the database is a Variant Dataset or Sequence Annotation Dataset. Click the tool icon in the Dataset section, name it, and select the dataset type, click OK, on the Manage Dataset window click Apply 8.4 Manage Variant Datasets With Alamut Genova all created & imported variants and their editing history can be viewed in the Samples page Sample Pages To open the samples page. o Click Variant, click View All Variants Samples Window

61 Creating a Variant 61 In the Samples window you can. o Explore all existing variants and variant datasets. o Add information to the datasets. o Classify variants via variant or patient data. o Add variants to the created dataset.

62 Protein Structure View

63 63 Protein Structure View 9 Protein Structure View Alamut Genova allows you to view the secondary and tertiary structure of a protein, in order to view the location of variants occurring in the structure and what effect it may have on it. 9.1 View from Transcript 3D structures are available if: 3D coordinates determined by X-ray diffraction or NMR are available in the Protein Data Bank. To view the 3D structure of the working protein if available in the Protein Data Bank, first select a transcript where it is active, or click on the spanner, View, click Add Track, select Protein Secondary Structure in your track selection. The Secondary Structures track will appear on the main page. o Right-Click on the track, select View*gene name*in 3D and the 3D Protein Structure Window will appear. 9.2 View from Variant To view the 3D protein from a variant. o Right-Click on a variant, select Create. The Information Tab in the Variant Panel will appear.

64 64 Protein Structure View At the bottom of the Protein Level box, the Protein Data Bank will show the protein selected. o Click View and The 3D Protein Structure Window will appear D Protein Structure Window On the 3D Protein Structure Window use your mouse to click and drag to navigate the structure.

65 65 Protein Structure View By default the structure is presented in a rainbow colouring scheme, you have the possibility to change the colour and take a screenshot.

66 Track Configuration and Settings

67 67 Track Configuration and Settings 10 Track Configuration and Settings In Alamut Genova variant data is displayed on different tracks, you can select the information you would like to shown on each track and you can import databases from your computer, in order to create new tracks. Track information can be assembled using the Track Configurations 68. Alamut Genova has three default track configurations on first launch: Default, Protein and Region. You can create your own track configurations and modify the default configurations Application Settings All modifications to tracks and track configurations are made on the Application Settings window on the View Tab. To access the Application Settings window: o Click on the Application Settings icon, select the View Tab.

68 68 Track Configuration and Settings Note: The Application Settings window includes the following tabs: Network, View, Laboratory Databases, Classification, Misc, and Patient for more information on each tab see Configuration (INSTALLATION/Installation/ Configuration) 10.2 Track Configuration On the View Tab on the Track Configuration scroll menu you can: o Create a New Track Configuration. o Delete a Track Configuration Creating a New Track Configuration To create a new track configuration. o Click on the New button and type name of your new track configuration, click OK. o Your new track configuration will be saved and available on the main page and on the Application Settings window 67. o This track configuration can be changed, updated or deleted bused on the users requirements Deleting a Track Configuration To delete a configuration (new or default) select the configuration you wish to delete from the Track Configuration 68 scroll menu and click the Delete button.

69 69 Track Configuration and Settings Switch Between Track Configurations Alamut Genova allows you to switch between track configurations whilst on the main page Modifying Tracks When you select a track configuration you can modify what tracks are included in it and what information is available on the tracks (when applicable) Add Track(s) To add a track from an existing database. o On the View tab, click Add Track. o The Select Track window will open, select a track from the list, click Add.

70 70 Track Configuration and Settings Selecting Information Select/Deselect the information you wish to included on a track when available. Click on the sub headings underneath the track name, click Apply, click OK. Modify Frequency Thresholds: When selecting the information you wish to display on a track you can modify the frequency thresholds, where applicable, for a given database.

71 71 Track Configuration and Settings Ordering Track(s) You can change the order in which the tracks are displayed, by clicking on the track name and dragging it up/down to place it where you wish Deleting Track(s) To delete a track from the track configuration, click on the track, and click Delete Track.

72 Tracks

73 73 Tracks 11 Tracks Alamut Genova displays created and imported variants, nucleotide annotations, imported databases and personally created databases on tracks. Alamut Genova now enables you to add a Splicing Predictions Track 84 and a Sanger Electropherogram 86 Track. o Allele Frequency Databases 73. o Classified Variant Databases 75. o Genes 76. o Genome 78. o Laboratory Variant Databases 79. o Nucleotide Conservation 79. o Orthologues 80. o Sequence Annotations 80. o Protein Domains 81. o Protein Secondary Structure 82. o Protein Variants 82. o Regulatory Regions 82. o Repeat Masker 83. o Somatic 83. o Structural Variants 84 : The Database of Genomic Variants (DGV) track now displays the Copy Number Variation (CNV) of the region on the track. o Splicing Predictions Track 84. o Sanger Electropherogram Track 86. o BAM Alignments Allele Frequency Databases The Allele Frequency Databases track displays variants from external variant databases:? 1000 Genomes? Danish2K? gnomad? ExAC? ESP? HGVD? GoNL? Swegen

74 74 Tracks? Kaviar? dbsnp? Wellderly Deletions and Deletion-Insertions are displayed as squares on the Del/Delins sub track. Insertions and Duplications are displayed as a pentagons, on the Ins/Dup sub track. Substitutions are displayed in a hexagon on the Subst sub track. To In the View Tab of the Application Settings window. you can select or unselect which database you wish to include in the track. Modify (when applicable) the frequency ranges of the databases on the track.

75 75 Tracks 11.2 Classified Variant Databases The Classified Variant Databases track displays information from: o Clinvar Display The color of the variant depends on the pathogenic classification given to the variant. o Del/Delins: Displays Deletions and Deletion/Insertions as rectangles. o Ins/Dup: Displays Insertions and Duplications variants as pentagons. o Subst: Displays Substitutions as hexagons. o By default the alternative allele is displayed. If various variants are reported with the same position then the allele number is reported (not the allele alternative). To annotate and/or modify variant information: click to select the variant, right-click and select Create. To view which external database the variant is from, right click on the variant and the databases will be listed in the pop up window.

76 76 Tracks In the Application Settings window you can apply a filter to Clinvar in order to view only the variants with a specified pathogenicity Genes The Genes track displays the genes of your selected transcript.

77 77 Tracks Nucleotide Position Hover mouse arrow over a nucleotide to view a pop up, showing: c.: Gene coordinate position. p: Protein position. g: Genomic position. Pos/ATG: Position of nucleotide from ATG position. Exon: Exon that contains the variant Transcript Options You can load another transcript for the selected gene, whilst on the Gene track. o Click on the transcript name tag and right-click to open the submenu. View All: When you select view all the list of transcripts will appear under the default one, you can select your desired transcript here. Set Preferred Transcript allows you to select the transcript you would like to make your default (preferred) transcript. When you select a transcript from the list it will be loaded automatically next time you search for the gene. View Transcript at NCBI RefSeq: Select to view the RefSeq transcript in the NCBI gene database. View Transcript in Ensembl: Select to view the Ensembl transcript in the Ensembl database. External Variants: Select to view PubMed Extracts showing available articles on variants for the gene you are studying.

78 78 Tracks Create Variant and Create Sequence Annotation Zoom in to view nucleotides. Right-Click on a nucleotide to. o Make Variant: to create a variant. o Make Region: to create a nucleic sequence annotation. o Copy: This function copies the highlighted nucleotide(s) in order for you to be able to paste the sequence elsewhere. o Copy Reverse/Complimentary: This tool will copy the selected nucleotide(s) and when you paste, the original sequence will appear followed by the reversecomplementary counterpart. o Copy Position: Select to copy the genomic position of your selected nucleotide(s). o Copy C Position: Select to copy the gene coordinate position along the track Genome The Genome track displays the available nucleotides. In order to determine the nucleotide position, the track name additionally included the chromosome genomic position.

79 79 Tracks Hover mouse arrow over a nucleotide it will show the genomic position 11.5 Laboratory Variant Databases The Laboratory Variant Databases track displays the imported variant databases or personally created variants. The Laboratory Variant Databases track will include the sub-tracks. o Del/Delins: Displays Deletions and Deletion-Insertions as rectangles. o Ins/Dup: Displays Insertions and Duplications as pentagons. o Subst: Displays substitutions as hexagons Nucleotide Conservation The Nucleotide Conservation track displays the nucleotide conservation scores from the databases PhastCons and PhyloP. o PhastCons scores are displayed in grey. o PhyloP scores are displayed in blue. o You can select to view the PhastCons only by right-clicking on the small settings icon and selecting Display PhastCons only.

80 80 Tracks 11.7 Orthologues The Orthologues track displays protein orthologue alignments from the Ensembl database, ICAR or IBC (In-house). These alignments show the conservation of amino acids across different species Sequence Annotations The Sequence Annotations track displays your personal annotations in rectangles along the track. To create a sequence annotation. o On the Gene or Genome track, select your desired nucleotides, right-click and select Make Private Annotation. o The Private Annotation window will launch. Complete the Sequence, Features section and External Link as necessary.

81 81 Tracks 11.9 Protein Domains The Protein Domains track will display the domain information from InterPro (via Ensembl).

82 82 Tracks Protein Secondary Structure The Protein Secondary Structure track displays the availability of a 3D Protein Structure. If a structure is available you will see alpha-helices in turquoise, the betastrands in bordeaux and the turns in green (the structure is aligned to where the protein structure overlaps the genome coordinates). Note: See Protein Structure View 63 for full information on launching the 3D protein structure window Protein Variant The Protein Variant track displays variant sets at protein level from PDB Swissvar. o You can see the amino acid information by hovering over a variant on the track Regulatory Regions The Regulatory Regions track displays a genome-wide set of regions that are likely to be involved in gene regulation. The track displays regulatory regions determined by databases. o Ensembl 76: TarBase, cisred, Ensembl Regulation, Vista Enhancer. o FANTOM5 Phase1. The track includes the sub-tracks. o cisred motif. o CTCF.

83 83 Tracks o Enhancer. o mirna target. o Open Chromatin. o Promoter. o Promoter Flanking Region. o TFBS. o TSS Repeat Masker The Repeat Masker track displays the repeat DNA regions identified by the Repeat Masker tool. The regions are displayed in accordance to their type. o SINEs (SINE). o LINEs (LINE). o DNA elements (DNA). o Simple Repetitions (Simple Repeat). o Low Complexity. o Satellite Elements (Satellite). o Small RNAs (RNA). o Other. o Not Classified (Unknown) Somatic The Somatic track displays information from COSMIC The track will display the sub-tracks: Deletion/Delins, Insertion/Duplication and Substitution in order to show what category the variant belongs to. In the Application Settings window you can modify the frequency score cut-off and select/unselect the status of variant pathogenicity displayed on the Somatic track.

84 84 Tracks Structural Variants The Structural Variants track will display the copy number variations in the genome via the DGV database Splicing Predictions The Splicing Predictions track will include the sub-tracks: Aggregate Score, Limnos, MaxEntScan, SSF, Gene Splicer and NNSplice. 49

85 85 Tracks In the Application Settings window you can select the frequency score cut-off for each of the mini tracks.

86 86 Tracks Sanger Electropherogram Track Alamut Genova allows you to upload Sanger Electropherogram (Sequencing) data and create a track. o Click File, click Open File and import your Sanger Electropherogram file A track will appear and display the Sanger Electropherogram data BAM Alignments Loading BAM Alignments To load BAM alignments to a track. o Click File, click Open BAM File and import your BAM alignments file.

87 87 Tracks BAM Track Display and Tools BAM Track Settings. o Show Reads: Select/Unselect to show reads on the track. o Show Coverage: Select/Unselect to show coverage on the track. o Variants. o Sequencing Targets. o Depth Coverage. o Reads BAM Track Settings Open the BAM Track Settings window to modify Base & Read Display, Load BAM & VCF Files and edit Preferences Base and Read Display Settings

88 88 Tracks Load BAM and VCF Files In order for the corresponding VCR target file to be loaded at the same time as the BAM file they must have the same name, otherwise you can load the corresponding VCF file manually. The loaded targets will appear on the sub-track Targets Preferences BAM Preferences Allows you to personalize the BAM track.

89 89 Tracks Targets: o Tick the Display box to display the targets on the BAM Track. o Number of intronic bases in exon-based targets: By default, sequencing targets are supposed to cover current gene s exons. This setting specifies the number of exonflanking intronic bases; to add to exon-defined targets. (Targets can otherwise be loaded from BED files using Load BED targets from files/ from URL). Coverage to display the thresholds on the BAM Track: o Linear/Quadratic Scale: the coverage histogram can either be displayed using a linear scale where the height of each bar is directly proportionate to the depth value, or using a quadratic scale where low depth values are increased and high depth values are decreased.

90 90 Tracks o Depth Threshold: Targets are highlighted in red where coverage depth is below the assigned threshold. Detected SNVs are only reported at positions where coverage is above the assigned threshold. o Allele Frequency Threshold: Here you can set the Allele Frequency thresholds. Single nucleotide variants (SNVs) that are detected will be displayed. (SNVs that are below the threshold will not be displayed). Display Size, select from these settings to define the graphical height of reads. Reads, to display the reads on the BAM Track: o Show all Bases: If Show all Bases is not checked, then read bases are displayed only if they differ from the reference sequence. o Filter PCR Duplicates: Alamut Genova does not itself detect PCR duplicates. Reads marked as PCR duplicates in the BAM file are withdrawn if this option is checked. o Max Displayed Read Depth: This setting only affects the graphical display of reads, not computations. o Mapping Quality Thresholds: Reads with a mapping quality under this threshold are withdrawn. o Shade Mismatched Bases by Quality: If this option is checked; bases with a Phred score under the specified minimum threshold will not be displayed. o Min. Phred Score & Max. Phred Score: Bases with a Phred score between the specified minimum and maximum thresholds are shaded, bases above the maximum threshold are displayed in full color. Insert Size: o Tick the Color read pairs, when insert size is out of bounds, if you want pair read distances to be highlighted in the BAM Track. o Fixed Bounds/Distribution-Based: According to insert size values provided in the BAM file, paired reads too distant or too close from each other are highlighted if this option is checked. Expected normal insert sizes can be expressed as fixed values or as a percentage over the distribution. If the insert size is large, denoting a deletion, reads are colored red. If the insert size is small, denoting an insertion, reads are colored blue. Scope: o Apply to this alignment only/apply to all currently open alignments.

91 Private Sequence Annotation

92 92 Private Sequence Annotation 12 Private Sequence Annotation With Alamut Genova you can create sequence annotations from nucleotide(s) you select. The annotations you create can be displayed on the tracks for future reference Annotate Nucleotide and Nucleotide Sequence Select your desired nucleotide(s), Right-Click and select Make Sequence Annotation. The Sequence Annotation window will appear. Here you can: name the region, select the database, dataset, colour, display and add any comments or external links. Click the Save (button) to finish and save.

93 93 Private Sequence Annotation Your sequence annotations will be displayed on the Sequence Annotations 80 track with your selected settings (colour, track position etc...). Click the Application Settings icon, Select View, click Add Track (button), and select Sequence Annotations. Note: This can additionally be used to store Laboratory Primers.

94 Data Source

95 95 Data Source 13 Data Source 13.1 Data Source List To access the data sources list from Alamut Genova. Click Help (on the toolbar), Data Sources. The Alamut Genova Data Sources window will appear with the list of data sources External Variant Databases Alamut Genova provides access to several databases of known variants. o Variants. Genome Aggregation Database gnomad 73. NCBI dbsnp 73. NHLBI GO Exome Sequencing Project ESP 73. The Genome of the Netherlands Consortium GoNL 73. The Human Genetic Variation Database HGVD 73 from The Japanese Genetic Variation Consortium. o Mutations. Swiss-Prot Variants SwissVar. COSMIC 83. NCBI ClinVar 75. o PubMed Extracts. o Variants and frequencies from the Whole-exome sequencing of 2,000 Danish individuals and the role of rare coding variants in type 2 diabetes PubMed. o Note: PubMed extracts are scanned and filtered by Interactive Biosoftware s Talamut engine External Variant Databases: Track Display All available variants, from the external databases, will be displayed by default on the tracks: Allele Frequency Databases Somatic , Classified Variant Databases 75 and External Variant Databases: Access The Variant External Databases are accessible by selecting a variant on these tracks: Allele Frequency Databases 73, Classified Variant Databases 75 and Somatic 83. To access the database(s): Click on variant (to select it), right-click, the submenu will appear.

96 96 Data Source o The submenu lists the external databases where the selected variant appears. Select your desired External Database: References Database of Single Nucleotide Polymorphisms (dbsnp). Bethesda (MD): National Center for Biotechnology Information, National Library of Medicine. Available from: Ensembl Variation database 96. Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA (URL: 96 ). Genome Aggregation Database (gnomad), The Broad Institute (URL: 96 ). The Japan Human Genetic Variation Database (HGVD), Japanese genetic variation consortium (URL: 96 ). The Genome of the Netherlands Consortium. Whole-genome sequence variation, population structure and demographic history of the Dutch population. Nature Genetics (2014) doi: /ng (URL: 96 ). Whole-exome sequencing of 2,000 Danish individuals and the role of rare coding variants in type 2 diabetes. Am J Hum Genet Dec 5;93(6): doi: /j.ajhg Epub 2013 Nov ClinVar: public archive of interpretations of clinically relevant variants.