Interactive Biosoftware offers a high-throughput annotation application named Alamut Batch. Alamut Batch is able to efficiently handle tens of thousands of variants. It is available as a client/server version for Windows and Linux, or as a standalone version for Linux only and it can be easily integrated into analysis pipelines. Alamut Batch is a high-throughput gene-based variant annotation application for Next-Generation Sequencing analyses ranging from gene panels to exomes. Designed for clinical and research laboratories, Alamut Batch enriches raw variants with a wealth of reliable and regularly updated attributes: they include effects on human genes, RefSeq transcripts and LRG sequences, accurate HGVS nomenclature descriptions, allele frequencies from different public databases such as 1000Genomes and ExAC, annotations on phenotypes from ClinVAR, COSMIC and HGMD Pro and on biological processes with Gene Ontology annotations. Alamut Batch computes predictions to assess the functional consequences of missense variants thanks to SIFT and Align GVGD. Alamut Batch also provides advanced splicing effect predictions based on MaxEntScan, HSF and other splicing prediction tools. You first need to download and install the program. Alamut Batch offers two different approaches for Windows users: a graphical user interface frontend and a command line program. If you want to use the graphical user interface, please select the corresponding application and double click on it. When you launch the Alamut Batch interface, you first need to browse and select the input file you want to annotate. Alamut Batch takes as input a list of genomic variations and outputs a list of annotations for each variant, when it is located inside a human gene locus. VCF files and tab-delimited files are supported for variant input. You can find more information on the format of these input files in the user manual. Once you’ve selected your input file, define the output files full path names in the Output section. By default, output files are saved in the Alamut Batch installation directory. The “Annotation file” is the annotation output file with annotated variants. If the “Failed variants file” is set, this file lists the variants that could not be annotated by Alamut Batch. In case you want to restrict annotation to specific variants, Alamut Batch allows you to input additional files such as a list of genes, transcripts or regions of interest. For example, let’s work on specific transcripts of 3 genes, BRCA1, BRCA2 and TP53. We need to create text files as follows and upload them into Alamut Batch. Alamut Batch also allows you to integrate external annotations in the annotation output file. More details about these files are available in the user manual. For the annotation settings, go to the Annotation tab and select the assembly your variants were called on. By default, Alamut Batch annotates variants on the longest transcript available in the Alamut database. You can choose to annotate all transcripts available or to annotate specific transcripts only. Missense and some splicing predictions can be computed optionally. The output tab allows you to specify options related to the output file. When using a VCF file as input, Alamut Batch can extract any information that you specify such as quality, read depth or genotype fields. If you have an HGMD Professional license, you can set Alamut Batch to get variant annotations from the HGMD database. Click on the “options” button, enter your HGMD credentials, click on “Ok” and restart the application. Click on “Run” to launch the analysis. Alamut Batch will let you know when the annotation process is complete. Here, you can visualize at any time the progress of the analysis process. Alamut Batch is also available through a Windows or Linux command line. It offers you the opportunity to combine Alamut Batch with other analysis tools for NGS into a pipeline to automate your workflow. When you want to use the command line tool, open a command prompt, go to the installation directory of Alamut Batch and execute Alamut Batch with the following parameters: the path of your input and output files which are mandatory and additional options such as “–alltrans” if you want to annotate your variants on all transcripts. More command line options are described in the user manual. Hit the return key.The analysis is launched. When it is complete, annotations will be available in the output tab-delimited file. Let’s now take a closer look at the output annotation file which is identical in both versions. It is a tab-delimited file with variants in rows and annotations in columns. Annotations include for example, allele frequencies from different public databases such as 1000 Genomes and ExAC projects, ClinVAR, HGMD and COSMIC, missense predictions such as SIFT or Align GVGD or splicing predictions from different tools such as MaxEntScan or HSF. All annotations are documented in the user manual. In order to use Alamut Batch to its full potential, we strongly recommend reading the user manual which is located in the Alamut Batch folder. If you have any questions regarding the use of Alamut Batch, you can contact our support team by email. Do not hesitate to visit our website to request a free 30-day trial of Alamut Batch. If you are interested in filtering the variants annotated with Alamut Batch, please take a look at the video presenting our variant filtration application Alamut Focus.