Abstract
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.
Highlights
Identification of somatic variants by next-generation sequencing has become an important technique in cancer research by pinpointing the genomic causes of tumour phenotypes
We have focused on exploring the general link between tumour sample cross-contamination and suspected contaminant variants of germline origin, but the effects of index misassignment require further consideration (Table 1)
Cross-sample contamination by recurrent somatic variants would represent a class of potential false positives that would be more difficult to recognize, and may have stronger clinical implications
Summary
Identification of somatic variants by next-generation sequencing has become an important technique in cancer research by pinpointing the genomic causes of tumour phenotypes. Our analysis of high-coverage tumour sequencing data shows that index misassignment is a source of false positive somatic variant calls in a form of true variation obtained from co-multiplexed samples. Consortium, Institute for Cancer Research, The Norwegian Radium Hospital/Oslo University Hospital, Oslo, Norway.
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