Spike-in panels

The Illumina FFPE DNA Prep with Exome 2.5 Enrichment supports the addition of probes during the enrichment process. Spike-in probes are intended to increase coverage in Exome v2.5 regions or add coverage in areas of no coverage by the Exome 2.5 panel. The analysis workflow will be similar, but attention should be paid to the resource files.

Systematic noise file. If the spike-in panel adds coverage to new regions of the genome, it is recommended to generate a new systematic noise file from normal samples enriched with Exome v2.5 plus spike-in. Otherwise, variants calls in the new regions are more vulnerable to false positive calls than regions where, based on the systematic noise file, a somatic filter tackles noise that consistently appears at specific locations in the reference genome.

Panel of Normals (PON) for CNV calls. Whether the spike-in probes adds coverage to new regions of the genome or increases coverage of coverage regions, it is always recommended to generate a custom PON, for your specific conditions, if calling CNVs. CNVs are called in regions characterized in the PON, so if the case sample has coverage in additional regions compared to the PON because of a spike-in, analysis will not call CNV events in those additional regions. However, the addition of spike-in probes can change enrichment broadly and may cause coverage fluctuations in previously characterized regions. Thus, CNV calls in regions that are shared by the PON and the case sample could be impacted by the addition of spike-in probes.