VariantStudio FAQs

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  • General

  • Yes, but only after validating the software according to Institution, Local, State, and Federal guidelines before using the Illumina VariantStudio software.

    VariantStudio needs external connections to annotate. For annotation, VariantStudio needs access to the annotation URL via port 80. This URL can be found in the installation folder, in the file VariantStudio.exe.config, next to the key annotationServiceUri. This link should be an Amazon Web Services or Illumina link. In addition to the annotation connection, VariantStudio must make a one-time connection to and, also through port 80. This connection serves to make sure that the user has access to the Illumina Annotation Service. This connection is needed again if the Forget BaseSpace Logon option is used, or if the BaseSpace account information is not saved.

    No. Illumina VariantStudio is a point-and-click software application that enables variant data exploration, annotation, and filtering without requiring bioinformatics expertise.

    Illumina VariantStudio v2.1 requires an internet connection for annotating variants because the annotation database, known as the Illumina Annotation Service, resides in Amazon Web Services. After variants are annotated and saved in a project, an internet connection is not necessary.

  • Workflow

  • Yes. Sample-to-sample comparison is done through the Cross Sample Subtraction Filter. This process filters variants that are present in one sample but not the other.

    If a new version of the Illumina Annotation Engine is released at the same time as a new software version release, the updates to the annotation database are specified in the software release notes. Otherwise, you receive an email notification informing you that a new database version is available. The email outlines the changes made relative to the previous annotation database version.

    By default, the Classification Database is saved locally in C:\ProgramData\Illumina\Illumina VariantStudio\ClasificationDb.bin. When the database is stored locally, classifications are available for the current project and any future projects opened on that computer.

    The Classification Database can also be configured for storage on a user-specified network location. When the database is stored on a network location, classifications are available to projects opened in any installation of VariantStudio with access to the network location.

    Yes. You can import custom annotations to the software using the Custom Annotation function in VariantStudio. The custom annotation file must be in tab-delimited text format (TSV) and must contain a header row that specifies the column names. VariantStudio allows both custom variant annotations and custom gene annotations.

    Yes. When generating a report from the Sample Report menu, you can either type in the sample information in the chosen report template or import the sample information from an external text file.

    No, you perform variant classification. With VariantStudio, you can provide annotations that can be used to classify variants within VariantStudio. After you assign a classification category to a variant, the information is saved to the Classification Database so that it can be applied to the same variant observed in other samples. Classifications saved in the Classification Database can be applied automatically to other samples through the Apply Classifications from Database menu.

    When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into two lines and both variants are annotated.

    Illumina uses BaseSpace authentication to make sure that you are authorized to use the application. After you have authenticated for the first time, VariantStudio saves your credentials so that you are not asked to log in again later. You can delete your credentials using Annotation & Classification | Annotation Options | Forget BaseSpace Logon.

    Illumina does not have a specific schedule for updating the annotation database. The update process largely depends on the cadence of updates from the different sources aggregated to create the annotation database.

    The desktop version of Illumina VariantStudio stores data locally in the VariantStudio project file. During the import of VCF files, data are loaded to the desktop memory. To save the data, designate a location to save the project. The project can be reopened later.

    The Illumina VariantStudio software uses multiple annotation sources. For more information, see the VariantStudio User Guide.

  • File Formats

  • Yes. VariantStudio v2.1 and v2.2 support VCF and gVCF file formats. For more information, see the VariantStudio User Guide.

    Importing large gVCF files requires more memory and takes longer to import. Use the following information as a guideline to estimate required RAM. This information is based on a Quad-Core Xeon® processor with 16 GB RAM.

    • Exome: 500-800 MB RAM per annotated exome
    • Whole genome: 3.5 GB RAM per annotated whole genome, 4 GB RAM recommended in addition to system memory. For example, if you are importing three annotated whole genome files, you need at least 12 GB but 16 GB is recommended to account for system memory.
    • Whole genome gVCF without hom-ref positions: 4.0 GB RAM per annotated whole genome in addition to system memory. VariantStudio imports non-hom-ref positions but it takes longer than 6 hours to go through all 300-400 million lines in the gVCF file. If you are not using the hom-ref import option, Illumina recommends using standard VCF files.
    • Whole genome gVCF with hom-ref positions: VariantStudio populates a maximum of 10 million rows in the Variants table. Therefore, it is not possible to import all positions. When all 10 million lines are populated, > 5 GB RAM without annotation is required. With annotation, 6-7 GB RAM is required. Alternatively, you can pre-process gVCF files for regions of interest before importing to VariantStudio.

    In VariantStudio, you can export information displayed in the Variants table, which includes variants and associated annotations in TSV file format. Sample reports are generated in either RTF or PDF file formats.

  • Compatibility

  • No. If samples are reannotated in VariantStudio v2.2, all annotations for variants are replaced with the annotations from the newer version of the Illumina Annotation Service. If you do not want to lose previous annotations for these samples, create a project for the samples that you want to preserve with the old annotations.

    Yes. VariantStudio v2.1 and v2.2 can be installed concurrently on the same computer. The two versions of VariantStudio operate independently; information is saved in different application spaces. VariantStudio v2.1 installs as "Illumina VariantStudio 2.1", while v2.2 installs as "Illumina VariantStudio 2.2" and does not overwrite the v2.1 installation.

    Using the default installation folder for both versions guarantees concurrent usability. If you decide to define a custom installation path, do not use the same installation folder for both versions.

    No. VariantStudio is tested and supported on Windows 7 or later. It is possible to use virtualization software such as Parallels or VMware to run a Windows application on a Mac or Linux. However, this method is an unsupported function. If you choose to run VariantStudio using a virtual system, allocate at least 2 GB of RAM to the system and close any other programs.

    Yes. The Amplicon-DS workflow in MiSeq Reporter software is used for the alignment and variant calling of TruSight Tumor data. The resulting VCF files can be imported into VariantStudio. Illumina recommends that you use the merged VCF file from the Amplicon-DS workflow. In these files, the variant call information from both the forward pool and reverse pool has been merged.

    Yes. However, if analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the columns required by VariantStudio. See the VariantStudio User Guide (document # 15040890) for information about VCF file requirements.

    Illumina supports the previous version of VariantStudio software for 12 months after a new version is released.

    The content of the Annotation Database is aggregated from a broad range of external sources, including public and private sources. At the time of a release for the Annotation Database, the content is locked down and versioned.

    Due to the nature of private annotation sources, our ability to maintain annotation content for the 12-month period might not be possible. In these rare occurrences, Illumina communicates the changes as soon as possible to reduce any potential impact on your pipeline.

    No. The Illumina VariantStudio End User License Agreement (EULA) states that VariantStudio must be used solely to analyze data generated from an Illumina sequencing instrument.