How to use Exome 2.5 Panel of Normals reference for germline deletion/duplication analysis

For whole-exome sequencing, the DRAGEN Copy Number Variant (CNV) pipeline supports a reference-based normalization algorithm that uses several reference samples to determine a baseline level from which to call CNV events. The reference used for this algorithm is referred to as a Panel of Normals (PON). The reference samples have undergone the same library prep, sequencing, and data processing workflow as the case sample. This allows the algorithm to subtract out system-level biases that are not sample specific.

A default Panel of Normals reference is available to evaluate germline CNV calling as part of the Illumina Exome 2.5 (formerly named, 2.0 Plus) sequencing workflow.

Panel of Normals reference built from NovaSeq 6000 sequencing data

From an ancestrally diverse set of reference samples, at least 50 ng of genomic DNA was used to prepare libraries with Illumina DNA Prep for Exome 2.5 Enrichment following Illumina recommended protocols and overnight hybridization. Libraries were sequenced on NovaSeq 6000 and processed with the DRAGEN CNV calling pipeline using default settings.

Where to access the Panel of Normals reference

The PON reference can be accessed for download on the Illumina Support site here or in the DRAGEN Enrichment bundle in Illumina Connected Analytics (ICA).

When to use the Panel of Normals reference

This PON may be used to evaluate deletion/duplication analysis of samples prepared with Illumina DNA Prep for Exome 2.5 Enrichment and sequenced with the NovaSeq 6000 or platform. Use of this PON with sequencing data from other platforms will need to be validated by the user.

How to use the Panel of Normals reference

• For DRAGEN Enrichment App: During run setup, enable CNV calling and upload the Panel of Normals *.gz file where indicated.

• For DRAGEN on-premise server: Command line support for whole-exome normalization can be found on the Illumina Support site here.

• For analysis in Emedgene: Contact the local Illumina sales representative or [e-mail] to request deployment of the default Exome 2.5 PON and kit in the relevant Emedgene account.

For any feedback or questions regarding this article (Illumina Knowledge Article #7918), contact Illumina Technical Support [e-mail].