Alamut® Focus: How to use the software

This page is the written transcript of the video tutorial entitled "Alamut® Focus: How to use the software", available here

Interactive Biosoftware offers an interactive variant filtration application for NGS analysis to help users identify candidate genetic variations. Designed for clinical and research laboratories, Alamut Focus facilitates the filtration of genetic variants from a group of individuals related or not, one of them at least being affected by a genetic disorder. Starting from annotated variant collections, Alamut Focus allows you to quickly design and apply simple to complex variant filtering strategies, based on user-defined criteria or pre-configured ones. It allows you to identify causal variants annotated by Alamut Batch, the high-throughput annotation application that is able to handle hundred thousands of variants.

You first need to download and install the program. Launch the application by double clicking on it.

The first time you launch Alamut Focus, you need to import your annotated variants through the "File" menu and select one or several files.

Selected files are automatically imported and managed as datasets in the Alamut Focus database. Next time you launch the application, your previous datasets will be directly available for filtering.

Let's open a dataset and start to design a variant filtering strategy. Click on "Open Dataset" and select it. It will appear in the main window of Alamut Focus that is split in two sections: a "Filter Panel" at the top providing filtering criteria and a "Variants Table" at the bottom displaying annotated variants.

The "Filter panel" provides an easy way to apply any combination of filters by navigating through several tabs.

On the right-hand side, a "Filter Summary" automatically displays all filters applied on your data and the number of filtered variants. Filters are applied as soon as they're selected.

The "Variant Table" displays annotated filtered variants that you can export for further analysis.

Let's now take a closer look at filters available in Alamut Focus. The first tab named "General" is used to specify the variant Type and its chromosome location. You may also specify the Variant Frequency and the Genotype, which are sample-related properties.

The "Gene" tab allows you to include or exclude specific genes and specify variant locations within gene loci. You can also use the Gene Ontology project annotations to filter your dataset.

Variants having a coding effect or a known clinical impact relative to phenotypes described in dbSNP, ClinVar, HGMD Pro or Cosmic databases can be pointed out through the "Consequence" tab.

The "In Silico Prediction" tab allows a filtering strategy based on predictions of missense and/or modified splicing effects. For instance, if you want to explore variants that have a repressive effect on their nearest natural splice site, you can apply the "Repressed" filter combined to specific prediction scores.

The "Population Frequency" tab allows you to refine your dataset according to their relative frequencies among specific populations extracted from the 1000 Genomes Project or ExAC for instance. Filters can be applied to the global population or subpopulations for each source. For the dnSNP database, you also have the possibility to filter variants according to their validation status.

"Quality" filters are also available to filter variants on quality criteria such as Depth of coverage or variant calling quality.

Finally, variants consistent with an inheritance mode can be pointed out with the "Study Design" filter. This filter is composed of two parts: The Description allows you to qualify selected datasets in the study context; the Inheritance Mode allows you to define how a particular genetic trait or disorder is passed from one generation to the next.

Alamut Focus enables you to manage and reuse filters and projects to speed up your analyses.

To save combinations of filters and use them later on other datasets, use the "Save Filter as..." option through the "Filter" menu.

To apply a previously saved Filter, go to "Filter>Apply" and select a filter name from the list. It will be automatically applied to the current dataset.

The filtered variant subset and its related filters can be saved using the Project menu.

Alamut Focus allows you to apply annotations and classify variants according to their biological impact. To do so let's go to the Variant Tab. Right click on a variant in the "Variant Table" and select "Edit Variant". The Variant Tab displays all relevant information about the selected variant.

The "Information" section displays genomic and gene-related descriptions and shortcuts to external tools such as Alamut® Visual, OMIM, and HGMD. The "Pathogenicity" section enables you to classify your variant. Note that pathogenicity class is not automatically computed. Additional information can be filled in the "Comments" text area. "Same variants" displays other datasets in which the variant is also found. Click on Save to store annotations. Once you've saved annotations, you can see in the "Variant history" all changes that have been made to the variant classification.

You can export filtered variants and their annotations from the Variant table by clicking here. It will generate a tab-separated file. You can also generate an Alamut Visual Mutation file for each gene. Afterwards, this will allow you to import variants into Alamut Visual.

In order to use Alamut Focus to its full potential, we strongly recommend you to read the documentation. To do this, go to the menu "Help > Documentation".

If you have any questions regarding the use of Alamut Focus, you can contact our support team by email via the menu "Help"/"Contact Support".

Don't hesitate to visit our website to request a free 30-day trial of Alamut Focus.

If you are interested in visualizing and interpreting filtered variants from Alamut Focus, please take a look at the videos presenting the graphical variant interpretation application Alamut Visual here.