Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust high-throughput sequencing. Index misassignment leading to mutual contamination between pooled samples has however been described as a general problem of the latest Illumina sequencing instruments utilizing exclusion amplification. Using real-life data from multiple tumour sequencing projects, we demonstrate that index misassignment can induce artefactual variant calls closely resembling true, high-quality somatic variants. These artefactual calls potentially impact cancer applications utilizing low allelic frequencies, such as in clonal analysis of tumours. We discuss the available countermeasures with an emphasis on improved library indexing methods, and provide software that can assist in the identification of variants that may be consequences of index misassignment.