a lam ut g e nov us r m - interactive-biosoftware.com · alamut ® genova is a component of the...

®

Alamut Genova User Manual(EN) Version 1.4

USER MANUAL

© 2018 Interactive Biosoftware, a SOPHiA GENETICS CompanyLast modified date: 3rd of December 2018

2

Alamut Genova User Manual (EN) Version 1.4 © 2018 Interactive Biosoftware, a SOPHiA GENETICS Company

Table of Contents

1. Preamble 6

1.1 Contact Us ............................................................................................................................ 7

2. Enhanced Features 8

3. Introduction 10

3.1 Prerequisites ...................................................................................................................... 11

Operating System ........................................................................................................ 113.1.1

Internet Connection ..................................................................................................... 113.1.2

Hardware Requirements ............................................................................................. 113.1.3

3.2 License Type ....................................................................................................................... 12

3.3 Installation Step-by-Step .................................................................................................. 12

3.4 Configuration ..................................................................................................................... 15

License ........................................................................................................................ 163.4.1

Network ....................................................................................................................... 193.4.2

View ............................................................................................................................. 203.4.3

Laboratory Variant Databases .................................................................................... 213.4.4

Classification ............................................................................................................... 223.4.5

Misc ............................................................................................................................. 233.4.6

Patient ......................................................................................................................... 243.4.7

Report .......................................................................................................................... 253.4.8

3.5 Homepage Overview ......................................................................................................... 26

3.6 Interface ............................................................................................................................. 26

Interface: Shortcut Buttons ......................................................................................... 263.6.1

Interface: Menu Options ............................................................................................. 273.6.2Applications ................................................................................................................................................... 283.6.2.1

File ................................................................................................................................................................... 283.6.2.2

View ................................................................................................................................................................. 293.6.2.3

Web .................................................................................................................................................................. 303.6.2.4

Variant ............................................................................................................................................................ 303.6.2.5

Tools ................................................................................................................................................................ 313.6.2.6

Help .................................................................................................................................................................. 313.6.2.7

3.7 Reported Problems and Solutions ................................................................................... 31

4. Genomic View 33

5. Chromosome View 35

5.1 Overview of Chromosome ................................................................................................ 36

5.2 Overview of Transcript ..................................................................................................... 36

5.3 Select a Transcript ............................................................................................................. 37

3


Table of Contents

6. Direct Gene Search 38

7. Variant View 41

7.1 Main Variant Window and Variant Display .................................................................... 42

Navigation ................................................................................................................... 427.1.1

8. Creating a Variant 43

8.1 Variant Properties ............................................................................................................. 44

8.2 Variant Panel ..................................................................................................................... 45

Information Tab ........................................................................................................... 458.2.1

Annotation Tab ............................................................................................................ 468.2.2

Splicing Tab ................................................................................................................. 498.2.3Splicing Tab Display ..................................................................................................................................... 508.2.3.1

Splicing Tool Menu ....................................................................................................................................... 518.2.3.2

ESP Prediction Tab .................................................................................................................................. 518.2.3.2.1

Options ..................................................................................................................................................... 528.2.3.2.2

Highlight Differences .............................................................................................................................. 538.2.3.2.3

Report ........................................................................................................................................................ 538.2.3.2.4

Splicing Track ................................................................................................................................................ 548.2.3.3

Background on Prediction Methods .......................................................................................................... 548.2.3.4

Set of Human Constitutive Exon/Intron Junctions ............................................................................ 568.2.3.4.1

References ................................................................................................................................................ 578.2.3.4.2

Report Tab ................................................................................................................... 588.2.4

8.3 Import and Create a Variant Database ............................................................................ 59

Import .......................................................................................................................... 598.3.1

Create .......................................................................................................................... 608.3.2

8.4 Manage Variant Datasets ................................................................................................. 60

Sample Pages .............................................................................................................. 608.4.1

9. Protein Structure View 62

9.1 View from Transcript ........................................................................................................ 63

9.2 View from Variant ............................................................................................................. 63

9.3 3D Protein Structure Window ......................................................................................... 64

10. Track Configuration and Settings 66

10.1 Application Settings .......................................................................................................... 67

10.2 Track Configuration .......................................................................................................... 68

Creating a New Track Configuration ........................................................................... 6810.2.1

Deleting a Track Configuration ................................................................................... 6810.2.2

Switch Between Track Configurations ........................................................................ 6910.2.3

4


Table of Contents

10.3 Modifying Tracks ............................................................................................................... 69

Add Track(s) ................................................................................................................ 6910.3.1

Selecting Information .................................................................................................. 7010.3.2

Ordering Track(s) ........................................................................................................ 7110.3.3

Deleting Track(s) ......................................................................................................... 7110.3.4

11. Tracks 72

11.1 Allele Frequency Databases .............................................................................................. 73

11.2 Classified Variant Databases ............................................................................................. 75

Display ......................................................................................................................... 7511.2.1

11.3 Genes .................................................................................................................................. 76

Nucleotide Position ..................................................................................................... 7711.3.1

Transcript Options ....................................................................................................... 7711.3.2

Create Variant and Create Sequence Annotation ....................................................... 7811.3.3

11.4 Genome .............................................................................................................................. 78

11.5 Laboratory Variant Databases ......................................................................................... 79

11.6 Nucleotide Conservation .................................................................................................. 79

11.7 Orthologues ....................................................................................................................... 80

11.8 Sequence Annotations ..................................................................................................... 80

11.9 Protein Domains ................................................................................................................ 81

11.10 Protein Secondary Structure ........................................................................................... 82

11.11 Protein Variant .................................................................................................................. 82

11.12 Regulatory Regions ........................................................................................................... 82

11.13 Repeat Masker ................................................................................................................... 83

11.14 Somatic .............................................................................................................................. 83

11.15 Structural Variants ............................................................................................................ 84

11.16 Splicing Predictions ........................................................................................................... 84

11.17 Sanger Electropherogram Track ...................................................................................... 86

11.18 BAM Alignments ................................................................................................................ 86

Loading BAM Alignments ............................................................................................ 8611.18.1

BAM Track Display and Tools ..................................................................................... 8711.18.2

BAM Track Settings ..................................................................................................... 8711.18.3Base and Read Display Settings ................................................................................................................. 8711.18.3.1

Load BAM and VCF Files ............................................................................................................................... 8811.18.3.2

Preferences ..................................................................................................................................................... 8811.18.3.3

BAM Preferences ..................................................................................................................................... 8811.18.3.3.1

12. Private Sequence Annotation 91

5


Table of Contents

12.1 Annotate Nucleotide and Nucleotide Sequence ........................................................... 92

13. Data Source 94

13.1 Data Source List ................................................................................................................. 95

13.2 External Variant Databases .............................................................................................. 95

External Variant Databases: Track Display ................................................................. 9513.2.1

External Variant Databases: Access ........................................................................... 9513.2.2

References .................................................................................................................. 9613.2.3

14. Programatic Access 97

14.1 Programmatic Access For Software Providers ................................................................ 98

Index 0

Preamble

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 humangenetics professionals and with critical judgment. Interactive Biosoftware cannotguarantee 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 email to Technical Support team via the homepage of AlamutGenova:

· Click Help on the Alamut® Genova homepage menu, click Contact Support([email protected] ).

2. Or you can contact support directly using the following email address:[email protected].

7

mailto:[email protected]

Enhanced Features

9


Enhanced Features

2 Enhanced Features

New Features

· Genomic View .

· CNV in Structural Variants /Database of Genomic Variants (DGV): The Copy

Number Variation (CNV) appears in the Database of Genomic Variants (DGV) track.

Functions

· 3D Protein Structure .

· Sanger Electropherogram Track .

· We have improved the functionality and visual display of variant annotation by creating

the Variant Panel .

· On the Annotation Tab we have added: (Variant View ; Create Variant ; Variant

Panel ; Annotation ). o The Human Phenotype Ontology (HPO).

o Suggested ACMG/AMP Classification .

· Predictions Track You can now see splicing predictions on a track.

34

84

63

86

45

42 44

45 46

22

84

Introduction

11


Introduction

3 Introduction

3.1 Prerequisites

The installation of Alamut® Genova needs the following technical specifications.

3.1.1 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-extractablearchive (.exe) or compressed file (.zip).

· Mac OS X – Snow Leopard/Lion/Mountain Lion/Mavericks/Yosemite/El Capitan (Mac

OS X 10.6-10.7-10.8-10.9-10.10-10.11).

Note: For Windows 32 bit and Mac OS users not all features are available.

3.1.2 Internet Connection

An internet connection is required to connect to the Interactive Biosoftware’sdatabase server:· Connection to the following IP addresses are required: 210.147.42; 212.129.54.111;

142.4.207.172; 212.83.147.70;· The software handles connections through HTTP on port 80 and 443, optionally

through a proxy.

3.1.3 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 arerequired on the software directory to ensure continued functioning of the applicationand user parameters.

12


Introduction

3.2 License Type

Alamut® Genova is installed with a floating license. Floating licenses can be installed onmultiple computers, with a limited number of concurrent users and are managed via anextranet .

3.3 Installation Step-by-Step

· Alamut® Genova extranet page: http://extranet.interactive-biosoftware.com.

· 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 e-mail.

· 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 Passwordprovided.

http://extranet.interactive-biosoftware.com

13


Introduction

· Begin Installation

oClick Downloads, click Installer.

· Follow the installation instructions in accordance with your computer operating

system:

oWindows – 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: http://extranet.interactive-biosoftware.com.§ Installation.

· If you have downloaded the Installer: execute it and choose an installation folder

where you have write permissions.


14


Introduction

· If you have downloaded the Self-Extractable executable (.exe) file, simply

double-click the installer to launch the installation program and follow theinstructions 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.

oMac OS X – Snow Leopard/Lion/Mountain Lion/Mavericks/Yosemite/El Capitan

(Mac OS X 10.6-10.7-10.8-10.9-10.10-10.11)

§ Download the Alamut® Genova(.dmg) file from our extranet page:

http://extranet.interactive-biosoftware.com. 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 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-2.11.0.dmg"You then "insert" or "mount" the disk image into the machine by double-clickingthe file. Having done this, the disk image will appear as another device in theFinder.

§ 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 yourApplications folder) and run it. Copying the program is performed simply byusing 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 theinstallation process. This can be done by using the eject icon next to it in theFinder 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.

11


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 softwareruns effectively: License, Network, if applicable Misc (HGMD Credentials). SeeConfiguration .

3.4 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

· Network

· View

· Laboratory Variant Databases

· Classification

· Miscellaneous

· Patient

· Report

15

16

19

20

21

22

23

24

25

16


Introduction

3.4.1 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.

oUser 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


Introduction

§ Connect to our extranet page (https://extranet.interactive-biosoftware.com ),select the Downloads section and if available download the updated versionshown.

§ Or on your computer find the Alamut® Genova Installation Directory and click the

Updater.exe file. The Maintain Alamut Genova Installer will launch, here selectUpdate Components and follow the instructions.

· Each time the user logs-in they will have to enter their Username and Password. Thelog-in window also includes a warning for users "This software application is agenomic variant exploration system that does not provide recommendations formedical diagnostics. It must be used by human genetics professionals and withcritical judgment."

16

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


Introduction

3.4.2 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 10001 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.oUse Proxy: Tick the Use Proxy box to complete the necessary proxy details.

20


Introduction

3.4.3 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. SeeTrack Configuration & Settings .o Show Selected Transcript on Variant Tracks: select/deselect to view the selected

transcript on the tracks.oDescription of Stop Codons: Select from X or HGVS 2.0 standard nomenclature

· Default Genome Build.oGRCh37 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 you wish to modify. See

Track Configuration to view details on how to modify tracks and their configuration.

67

67

21


Introduction

3.4.4 Laboratory Variant Databases

· See Import and Create Variant Database .

oWhen 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 tocreate a database.

oWithin 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.oWhen 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


Introduction

3.4.5 Classification

· Pathogenicity Classes Scheme (ACMG/AMP like).

o Personalize the names of the pathogenic classifications.

23


Introduction

3.4.6 Misc

· Mouse Wheel: two mouse scroll options are available.

· Sanger : for your Sanger Sequencing data you can set your required Quality

Threshold.

86

24


Introduction

3.4.7 Patient

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

25


Introduction

3.4.8 Report

· In the Report Tab you can select what information you wish to include by default in avariant report .58

26


Introduction

3.5 Homepage Overview

· On the homepage there there are a number of Menu Options , a bar of ShortcutButtons and the Go to search bar.

· On the left-hand side of the homepage a number buttons to improve the userexperience 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 theuser such as: the version of Alamut Genova; last update date; license expiry date(Valid until); and the number of available genes.

· The following Interface 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 navigatethe Main Window and the options that are available from the Menu & Shortcut Buttons.

3.6.1 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

26

26

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 theselected strand.

§ Opens the Genetic Code window which displays the Standard Genetic Code and the Mitochondrial Genetic Code.

§ Opens the Amino Acid Comparison window.

oWeb 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.

oGo 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 beloaded on the tracks.

3.6.2 Interface: Menu Options

· Application, File, View, Web, Variant, Tools and Help.

28


Introduction

3.6.2.1 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.

3.6.2.2 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


Introduction

3.6.2.3 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 agene 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 theselected position).The distance between the two positions will be displayed on theruler. You can click and drag the A & B markers to select different positions.

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.

3.6.2.4 Web

· Select from the list to view a studied sequence or gene in an external database orviewer.

3.6.2.5 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


Introduction

3.6.2.6 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.

3.6.2.7 Help

· Software Documentation.

· Data Sources.

· License Agreement.

· Software References: how to cite Alamut® Genova in a publication.

· Contact Support: to send an email to our Customer Support team an email

([email protected]).

· 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.

mailto:[email protected]

32


Introduction

oA: 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.

oA: 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.

oA: 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.

oA: Ask your IT Administrator to check that the proxy size limit is high enough and that

proxy cache has been disabled.

Genomic View

34


Genomic View

4 Genomic View

· The Genomic View allows you to visualize all chromosomes including the

mitochondria, intergenic genomic regions, structural variants and extragenicregulatory regions. The Genome build you view is based on your default preferencesin 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.

Chromosome View

36


Chromosome View

5 Chromosome View

In this section we will delve into the different viewing options available within thechromosome view: Overview of Chromosome and Overview of Transcript .

5.1 Overview of Chromosome

· When you have selected a chromosome from the Genomic View 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 and reverse direction icons on the toolbar, in order to make it easier to study it.

5.2 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.

36 36

34

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 , 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 bothSystematic and Custom naming is available for this transcript.

76

Direct Gene Search

39


Direct Gene Search

6 Direct Gene Search

· The fastest way to access a transcript is through the Gene button on the toolbar.

· Click the Gene button 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 Email Gene Request: If a gene or transcript version is not available, send our team

an email request.

o The Transcript Selection widow will open. Alamut® Genova will highlight the

canonical transcript by default in the transcript list.

40


Direct Gene Search

· Tick the "Preferred Transcript" box to select your preferred transcript.

Variant View

42


Variant View

7 Variant View

With Alamut® Genova you can annotate existing variants and create new variants. Inthis 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 VariantPanel .

7.1 Main Variant Window and Variant Display

· A new tab will open each time you open a transcript or create a variant. The tab will benames after the transcript or variant created e.g. "MLH1 NM_..."

· You can open several tabs at the same time.

7.1.1 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).oMove 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 thewindow).

oMove 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:oMove 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.

45

Creating a Variant

44


Creating a Variant

8 Creating a Variant

· Alamut® Genova allows you to create a variant and add it into the database withannotations you would like to include.oOn 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:oMake Sequence Annotation: Select to annotate a nucleic sequence (See Private

Sequence Annotation ).oCopy: This function copies the highlighted nucleotide(s) to enable the user to paste

the sequence elsewhere.oCopy rev/comp: This tool will copy the selected nucleotide(s) and when you paste,

the original sequence will appear followed by the reverse-complementarycounterpart.

oCopy genomic position: Select to copy the genomic position of your selectednucleotide(s).

oCopy cDNA position: Select to copy the coordinate position along the track of yourselected nucleotide(s).

oCopy Protein position: Select to copy the protein position of your selectednucleotide(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.

92

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.

8.2.1 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, PublicDatabases and Missense Predictions.

46


Creating a Variant

8.2.2 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


Creating a Variant

· The Annotation tab contains:

oDatabase & Sample: Select where you wish to save your newly created or

annotated variant.oComment: 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 choiceand select the file you would like to export.

oRNA Analysis: Enter in free-text form RNA analysis notes.

oOther 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 editedhistory of the variant, particularly helpful when more than one user is using thesoftware.

oClassification: Select the pathogenic classification of the variant and as a new

feature, access the ACMG/AMP Standards and Guidelines tool to enable the userto 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 additionalinformation about the patient, their phenotype or environment, it is not possible tomake a definitive conclusion without verifying the results experimentally withapproved genetics tests.

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


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 annotationname, and will output the populated spreadsheet or word document in the destinationfile.

8.2.3 Splicing Tab

· Alamut® Genova includes a splicing module integrating a number of prediction

algorithms and splicing prediction data:

· Splicing Signals:

oMaxEntScan – Donor, acceptor

oGeneSplicer- Donor, acceptor

oNNSPLICE- Donor, acceptor

o Known Constitutive Signals- Donor, acceptor

o Splice Site Finder-like (SSF) – Donor, acceptor, branchpoint

50


Creating a Variant

oMercer et al. – high-confidence branchpoints

o Limnos- branchpoints

· Exonic Splicing Enhancers (ESE) binding site detection:

o ESEFinder

oRESCUE-ESE

o EX-SKIP

· The Splicing Tab displays reference (wild-type) and mutated sequences, and the

splicing predictions.

8.2.3.1 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 for3’(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 thebar itself.

51


Creating a Variant

· A Splicing Track has additionally been added to Alamut® Genova.

8.2.3.2 Splicing Tool Menu

· The Splicing Tab includes a Splicing Tool Menu with the following tool buttons:

o ESE Predictions .

oOptions .

oHighlight Differences .

oReport .

oCopy Snapshot.

8.2.3.2.1 ESP Prediction Tab

· To display the ESE Predictions Tab.

o The ESE hits from ESEFinder are displayed above each sequence, and RESCUE-

ESE hexamers are shown below the sequence.

54

51

52

53

53

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.

oOptions: Click to launch the Splicing Predictions Options window.

oHighlight Differences: Click to reveal differences between wild-type and mutated

scores.oReport: Click to generate a separate report of the ESE Predictions.

oCopy 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.

8.2.3.2.2 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


Creating a Variant

8.2.3.2.3 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.

8.2.3.2.4 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


Creating a Variant

8.2.3.3 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.

oClick 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 .

8.2.3.4 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) andacceptor 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

84

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 fullyintegrated 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 thedonor subset of human constitutive exon/itron junctions, and each occurrence of the 6-mers (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-confidencehuman branchpoints in >10,000 genes, thus providing a first map of splicingbranchpoints 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 fromthe 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 calculatesthe 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


Creating a Variant

8.2.3.4.1 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 wereextracted 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).http://biodev.hgen.pitt.edu/cgi-bin/enologos/enologos.cgi .56

57


Creating a Variant

8.2.3.4.2 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, 2011.

· Cartegni et al. ESEfinder: A web resource to identify exonic splicing enhancers .

Nucleic Acids Res (2003) vol. 31 (13) pp. 3568-71

· Fairbrother et al. Predictive identification of exonic splicing enhancers in human

genes .Science (2002) vol. 297 (5583) pp. 1007-13

· Hellen Splice Site Tools: A Comparative Analysis Report .

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. 2012 Aug;33(8):1228-38.

· Mercer at al. Genome-wide discovery of human splicing branchpoints.

Genome Res (2015) 25(2): 290-303.

· Pertea et al. GeneSplicer: a new computational method for splice site prediction .

Nucleic Acids Res (2001) vol. 29 (5) pp. 1185-90

57

57

57

57

57

57

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 BRCA1exon 6. .Hum Mutat. (2011), 32, 436-444.

· Reese et al. Improved Splice Site Detection in Genie .

J Comp Biol (1997) vol. 4 (3), pp. 311-23

· 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): 7155-7174.

· Yeo et al. Maximum entropy modeling of short sequence motifs with applications to

RNA splicing signals .J Comput Biol (2004) vol. 11 (2-3) pp. 377-94

· Zhang et al. Statistical features of human exons and their flanking regions .

Hum Mol Genet (1998) vol. 7 (5) pp. 919-32

8.2.4 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:

oHGVS.

o ACMG/AMP Guidelines.

o Additional Information.

o External Variants.

o Splicing.

o Splicing Screenshot.

o Protein Information.

o Annotations.

oOccurrences.

oOrthologues.

· If you wish you can generate and save the variant.

· By clicking the right mouse button you can save and print your report.

57

57

57

57

57

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 variantdatasets and/or sequence annotation datasets. You can create tracks that display theinformation you have created or imported.

8.3.1 Import

· To import a database from your device into Alamut® Genova.

oOpen the Application Settings window.

o Select the Laboratory Databases tab, click on the plus button, select the

database from your files, select the option of Variant dataset, (If the databaseincludes Variant and Sequence Annotation datasets select both).

Note: The imported file must be formatted as a VCF file.

67

21

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 track,

you can add the database to the tracks.oWithin 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.

8.3.2 Create

· To create a personal database open the Application Settings 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 beviewed in the Samples page.

8.4.1 Sample Pages

· To open the samples page.

oClick Variant , click View All Variants

Samples Window

21

67

61


Creating a Variant

· In the Samples window you can.

o Explore all existing variants and variant datasets.

o Add information to the datasets.

oClassify variants via variant or patient data.

o Add variants to the created dataset.

Protein Structure View

63



9 Protein Structure View

Alamut® Genova allows you to view the secondary and tertiary structure of a protein, inorder to view the location of variants occurring in the structure and what effect it mayhave 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 proteinif 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 inyour track selection.

· The Secondary Structures track will appear on the main page.

oRight-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.

oRight-Click on a variant, select Create.

· The Information Tab in the Variant Panel will appear.

64



· At the bottom of the Protein Level box, the Protein Data Bank will show the protein

selected. oClick View and The 3D Protein Structure Window will appear.

9.3 3D Protein Structure Window

· On the 3D Protein Structure Window use your mouse to click and drag to navigate the

structure.

65



· By default the structure is presented in a rainbow colouring scheme, you have the

possibility to change the colour and take a screenshot.

Track Configuration and Settings

67



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 fromyour computer, in order to create new tracks. Track information can be assembled using

the Track Configurations . Alamut® Genova has three default track configurations on

first launch: Default, Protein and Region. You can create your own track configurationsand modify the default configurations.

10.1 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:

oClick on the Application Settings icon , select the View Tab.

68

68



Note: The Application Settings window includes the following tabs: Network, View, LaboratoryDatabases, 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:

oCreate a New Track Configuration.

oDelete a Track Configuration.

10.2.1 Creating a New Track Configuration

· To create a new track configuration.

oClick 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 .o This track configuration can be changed, updated or deleted bused on the users

requirements.

10.2.2 Deleting a Track Configuration

· To delete a configuration (new or default) select the configuration you wish to delete

from the Track Configuration scroll menu and click the Delete button.

67

68

69



10.2.3 Switch Between Track Configurations

· Alamut® Genova allows you to switch between track configurations whilst on the mainpage.

10.3 Modifying Tracks

When you select a track configuration you can modify what tracks are included in it andwhat information is available on the tracks (when applicable).

10.3.1 Add Track(s)

· To add a track from an existing database.

oOn the View tab, click Add Track.

o The Select Track window will open, select a track from the list, click Add.

70



10.3.2 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 givendatabase.

71



10.3.3 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.

10.3.4 Deleting Track(s)

· To delete a track from the track configuration, click on the track, and click Delete

Track.

Tracks

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 and a

Sanger Electropherogram Track.o Allele Frequency Databases .

oClassified Variant Databases .

oGenes .

oGenome .

o Laboratory Variant Databases .

oNucleotide Conservation .

oOrthologues .

o Sequence Annotations .

o Protein Domains .

o Protein Secondary Structure .

o Protein Variants .

oRegulatory Regions .

oRepeat Masker .

o Somatic .

o Structural Variants : The Database of Genomic Variants (DGV) track now

displays the Copy Number Variation (CNV) of the region on the track.o Splicing Predictions Track .

o Sanger Electropherogram Track .

o BAM Alignments .

11.1 Allele Frequency Databases

The Allele Frequency Databases track displays variants from external variantdatabases:

?1000 Genomes ?Danish2K

?gnomAD ?ExAC

?ESP ?HGVD

?GoNL ?Swegen

84

86

73

75

76

78

79

79

80

80

81

82

82

82

83

83

84

84

86

86

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


Tracks

11.2 Classified Variant Databases

· The Classified Variant Databases track displays information from:

oClinvar.

11.2.1 Display

· The color of the variant depends on the pathogenic classification given to the variant.

·

oDel/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


Tracks

· In the Application Settings window you can apply a filter to Clinvar in order to view only

the variants with a specified pathogenicity.

11.3 Genes

· The Genes track displays the genes of your selected transcript.

77


Tracks

11.3.1 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.

.

11.3.2 Transcript Options

· You can load another transcript for the selected gene, whilst on the Gene track.

oClick 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 loadedautomatically 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


Tracks

11.3.3 Create Variant and Create Sequence Annotation

· Zoom in to view nucleotides.

· Right-Click on a nucleotide to.

oMake Variant: to create a variant.

oMake Region: to create a nucleic sequence annotation.

oCopy: This function copies the highlighted nucleotide(s) in order for you to be able to

paste the sequence elsewhere.oCopy Reverse/Complimentary: This tool will copy the selected nucleotide(s) and

when you paste, the original sequence will appear followed by the reverse-complementary counterpart.

oCopy Position: Select to copy the genomic position of your selected nucleotide(s).

oCopy C Position: Select to copy the gene coordinate position along the track.

11.4 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


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.

oDel/Delins: Displays Deletions and Deletion-Insertions as rectangles.

o Ins/Dup: Displays Insertions and Duplications as pentagons.

o Subst: Displays substitutions as hexagons.

11.6 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


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 aminoacids across different species.

11.8 Sequence Annotations

· The Sequence Annotations track displays your personal annotations in rectangles

along the track.

· To create a sequence annotation.

oOn 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


Tracks

11.9 Protein Domains

· The Protein Domains track will display the domain information from InterPro (via

Ensembl).

82


Tracks

11.10 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 beta-strands in bordeaux and the turns in green (the structure is aligned to where theprotein structure overlaps the genome coordinates).

Note: See Protein Structure View for full information on launching the 3D protein structure window.

11.11 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.

11.12 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.

oCTCF.

63

83


Tracks

o Enhancer.

omiRNA target.

oOpen Chromatin.

o Promoter.

o Promoter Flanking Region.

o TFBS.

o TSS.

11.13 Repeat Masker

· The Repeat Masker track displays the repeat DNA regions identified by the RepeatMasker tool.

· The regions are displayed in accordance to their type.o SINEs (SINE).o LINEs (LINE).oDNA elements (DNA).o Simple Repetitions (Simple Repeat).o Low Complexity. o Satellite Elements (Satellite). o Small RNAs (RNA).oOther.oNot Classified (Unknown).

11.14 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


Tracks

11.15 Structural Variants

· The Structural Variants track will display the copy number variations in the genome via

the DGV database.

11.16 Splicing Predictions

· The Splicing Predictions track will include the sub-tracks: Aggregate Score,

Limnos, MaxEntScan, SSF, Gene Splicer and NNSplice.

49

85


Tracks

· In the Application Settings window you can select the frequency score cut-off for each

of the mini tracks.

86


Tracks

11.17 Sanger Electropherogram Track

· Alamut® Genova allows you to upload Sanger Electropherogram (Sequencing) data

and create a track.oClick File, click Open File and import your Sanger Electropherogram file

· A track will appear and display the Sanger Electropherogram data.

11.18 BAM Alignments

11.18.1 Loading BAM Alignments

· To load BAM alignments to a track.

oClick File, click Open BAM File and import your BAM alignments file.

87


Tracks

11.18.2 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.

oDepth Coverage.

oReads.

11.18.3 BAM Track Settings

· Open the BAM Track Settings window to modify Base & Read Display, Load BAM &VCF Files and edit Preferences.

11.18.3.1 Base and Read Display Settings

88


Tracks

11.18.3.2 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 correspondingVCF file manually.

· The loaded targets will appear on the sub-track Targets.

11.18.3.3 Preferences

11.18.3.3.1 BAM Preferences

· Allows you to personalize the BAM track.

89


Tracks

· Targets:

o Tick the Display box to display the targets on the BAM Track.

oNumber 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 exon-flanking intronic bases; to add to exon-defined targets. (Targets can otherwise beloaded 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 depthvalues are decreased.

90


Tracks

oDepth Threshold: Targets are highlighted in red where coverage depth is below the

assigned threshold. Detected SNVs are only reported at positions where coverageis 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 thatare 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 ischecked.

oMax Displayed Read Depth: This setting only affects the graphical display of reads,

not computations.oMapping 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.oMin. Phred Score & Max. Phred Score: Bases with a Phred score between the

specified minimum and maximum thresholds are shaded, bases above themaximum 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 thisoption is checked. Expected normal insert sizes can be expressed as fixed valuesor as a percentage over the distribution. If the insert size is large, denoting adeletion, 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.

Private Sequence Annotation

92



12 Private Sequence Annotation

With Alamut® Genova you can create sequence annotations from nucleotide(s) youselect. The annotations you create can be displayed on the tracks for future reference.

12.1 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. Clickthe Save (button) to finish and save.

93



· Your sequence annotations will be displayed on the Sequence Annotations 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.

80

Data Source

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.

13.2 External Variant Databases

· Alamut® Genova provides access to several databases of known variants.

o Variants.

§ Genome Aggregation Database gnomAD . § NCBI dbSNP .§ NHLBI GO Exome Sequencing Project ESP .§ The Genome of the Netherlands Consortium GoNL .§ The Human Genetic Variation Database HGVD from The Japanese Genetic

Variation Consortium.

oMutations.

§ Swiss-Prot Variants SwissVar.§ COSMIC .§ NCBI ClinVar .

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.

13.2.1 External Variant Databases: Track Display

· All available variants, from the external databases, will be displayed by default on the

tracks: Allele Frequency Databases , Classified Variant Databases andSomatic .

13.2.2 External Variant Databases: Access

· The Variant External Databases are accessible by selecting a variant on these tracks:

Allele Frequency Databases , Classified Variant Databases and Somatic .

· To access the database(s): Click on variant (to select it), right-click, the submenu will

appear.

73

73

73

73

73

83

75

73 75

83

73 75 83

96


Data Source

o The submenu lists the external databases where the selected variant appears.

Select your desired External Database:

13.2.3 References

· Database of Single Nucleotide Polymorphisms (dbSNP). Bethesda (MD): National

Center for Biotechnology Information, National Library of Medicine. Availablefrom: https://www.ncbi.nlm.nih.gov/SNP/ .

· Ensembl Variation database .

· Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA

(URL: http://evs.gs.washington.edu/EVS/ ).

· Genome Aggregation Database (gnomAD), The Broad Institute

(URL: http://gnomad.broadinstitute.org ).

· The Japan Human Genetic Variation Database (HGVD), Japanese genetic variation

consortium (URL: http://www.genome.med.kyoto-u.ac.jp/SnpDB ).

· The Genome of the Netherlands Consortium. Whole-genome sequence variation,

population structure and demographic history of the Dutch population. NatureGenetics (2014) doi:10.1038/ng.3021. (URL: http://www.nlgenome.nl/ ).

· Whole-exome sequencing of 2,000 Danish individuals and the role of rare coding

variants in type 2 diabetes. Am J Hum Genet. 2013 Dec 5;93(6):1072-86. doi:10.1016/j.ajhg.2013.11.005. Epub 2013 Nov 27.

· ClinVar: public archive of interpretations of clinically relevant variants.

https://www.ncbi.nlm.nih.gov/clinvar/.

96

96

96

96

96

96 96

96

https://www.ncbi.nlm.nih.gov/clinvar/

Programatic Access

98


Programatic Access

14 Programatic Access

Alamut® Genova allows Extended Programmatic access, in a similar way to Almaut®

Visual, further allowing convenient access to gene information.

To use this feature, first check that the external software tool you are using enables such

communication with Alamut® Genova. Second, in the Options dialog box (menu

'Application' > 'Settings' > 'Network' tab), possibly change the port according to the

external software specifications. Then restart Alamut® Genova.

14.1 Programmatic Access For Software Providers

Alamut® Genova embeds a small local HTTP server. This server listens to local HTTP

GET and POST requests coming through the port that is specified in the Options dialogbox. The default port is set to 10000 but it can be changed to any available port, (thisrequires the user to set the appropriate port in the Alamut® Genova Settings dialogbox).

Two types of HTTP requests are processed by Alamut® Genova:· http://localhost:10000/version

Upon receiving this request, useful for testing purposes, Alamut® Genova outputs its

current version as plain text.· http://localhost:10000/show?request=MLH1:c.464T>G

This request asks Alamut® Genova to display any data available in the system (herevariant "MLH1:c.464T>G").

Any request that can be processed by the Go to: input field of Alamut® Genova can be

sent out with the "show" verb.

· Example in Java Script

The following examples can be embeded in a web page (for different reasons theymight not work with all web browsers).

For this to work Alamut® Genova must be running and with port set to 10000 in theOptions dialog box.

Example script for the GET 'version' request:

99


Programatic Access

<script type="text/javascript">function httpGetVersion() { var xmlHttp = null; xmlHttp = new XMLHttpRequest(); if (xmlHttp) { xmlHttp.open( "GET", "http://localhost:10000/version", true ); xmlHttp.send(null); xmlHttp.onreadystatechange=function() { if (xmlHttp.readyState==4) { alert("Response from Alamut® Visual:\n"+xmlHttp.response); } } }}</script>

Example HTML form using this script:

<form><input type="button" value='View' onclick='httpGetVersion();' /></form>

Or view this page's source code.

Script for the GET 'show' request:

<script type="text/javascript">function httpGetFocusOn(focus) { var xmlHttp = null; xmlHttp = new XMLHttpRequest(); if (xmlHttp) { xmlHttp.open( "GET", "http://localhost:10000/show?request="+focus, true ); xmlHttp.send(null); }}</script>


<form><input type="button" value='View' onclick='httpGetFocusOn("MLH1:c.464T>G");' /></form>

Or view this page's source.

Example script for the GET 'show' request to load first a gene and then load a BAM file:

100


Programatic Access

<script type="text/javascript">function httpGetLoadBam(gene,bamFile) { var xmlHttp = null; xmlHttp = new XMLHttpRequest(); var bamXmlHttp = null; bamXmlHttp = new XMLHttpRequest(); if (xmlHttp) { xmlHttp.open( "GET", "http://localhost:10000/show?request="+gene+"&synchronous=true", true ); xmlHttp.send(null); xmlHttp.onreadystatechange=function() { if (xmlHttp.readyState==4) { bamXmlHttp.open( "GET", "http://localhost:10000/show?request=BAM%3C"+bamFile, true ); bamXmlHttp.send(null); } } }}</script>


<form><input type="button" value='Load a BAM file' onclick='httpGetLoadBam("MLH1","http://rd-connect.interactive-biosoftware.com/BAM/example.bam");' /></form>

a lam ut g e nov us r m - interactive-biosoftware.com · alamut ® genova is a component of the...

Documents