SEQ platform lists variant annotations of the variants detected in a sample on analysis details page. Therefore, sometimes there may be more than one annotation for the same variant. Figure 1 shows two annotations of the same variant. The variants located on overlapping genes may have more than one annotation. 

Figure 1: A variant with more than one annotations

The geneset of a kit is defined by the kit producer and is composed of the genes the kit covers. The genomic region a kit covers is defined by its manifest file. However, the genes in the kit’s geneset may not always be included 100% in the manifest file. For instance, the kit producer may design the kit A to include exons 1,3 and 5 of the gene A123; however, may exclude exons 2 and 4. As a result, these genomic regions are not included in the manifest file. Gene A123 is included in the geneset of kit A even though it is not fully present in the manifest file.
On-target variants are the variants that are located within the regions defined in the manifest file. Therefore on-target variants are divided in two:
1) Within the geneset (On-geneset variants): These variants are both within the manifest file and the geneset. 
2) Outside of the geneset (Off-geneset variants): Even though these variants are within the genomic regions defined by the manifest file, they are annotated according to genes that are not included in the geneset. For instance a variant may be located on the overlapping region of genes A123 and B456, and therefore can be annotated according to both genes. However, if the gene B456 is not included in the geneset of the kit, the annotation cannot be listed unless the ‘Showing off-geneset variants’ parameter is activated as shown in Figure 2.

Figure 2: Showing and hiding off-target and off-geneset variants

The variants seen in Figure 3 belongs to a BRCA kit and the geneset is composed of BRCA1 and BRCA2 genes. However, the variant in the figure can be annotated both according to BRCA1 and also an overlapping gene, RPL21P4. Since RPL21P4 is not included in the BRCA geneset, in order to see the annotation according to RPL21P4, ‘Showing off-target variants’ parameter should be activated as seen in Figure 2. 

Figure 3: Annotation of the same variant, on-geneset and off-geneset 

Off-target variants are the variations that fall out of target regions defined by the manifest file of the sequencing kit. Hence, off-target variants can be classified in two.
1) Within the gene set: Even though these variants are included in the kit’s gene set, they are outside of the target regions defined by the manifest file.
2) Outside of the gene set: These are the variants that are not only included in the manifest file, but also in the gene set. These variants can be divided in two. 
a) Annotated according to a gene: These variants can be annotated according to a reference transcript of a gene. 
b) Not annotated according to a gene (NOGENE): These are the intergenic variants that cannot be annotated according to a gene.

Off-target variants should be taken into account while new gene association studies and trio analyses. By enabling or disabling “See off-target variants” option you can list or hide these variants.

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