How to evaluate the targeted gene amplification (TGA) for Infinium BeadChips with Enhanced PGx Content

Overview

The Infinium workflow includes a targeted gene amplification (TGA) step for the following PGx BeadChips:

• Global Diversity Array v1.0 + Enhanced PGx (GDA + ePGx)

• Global Screening Array v4.0 + Enhanced PGx (GSA v4 + ePGx)

• Global Clinical Research Array v1.0 + Enhanced PGx (GCRA + ePGx)

TGA is a PCR amplification for specific pharmacogenomic (PGx) content, which allows for pseudogene disambiguation. The PGx TGA is combined with the Infinium whole genome amplification (WGA) before the Fragment step. The combined sample is processed through the standard Infinium workflow from Fragment on.

The TGA reagents contain a probe that generates a sample-independent TGA positive control fragment. This control fragment binds to the TGA Control Probe on the array when the PGx TGA and WGA are properly executed, recombined, and processed in the Infinium assay. The TGA Control Probe should have a positive signal, observed as Norm R on the Y axis of the SNP graph in GenomeStudio 2.0. The performance expectation for the TGA Control Probe (TGA_Ctrl_5716) is R > 1.

Table 1: PGx BeadChip Product specifications

BeadChip	Call Rate Specification	Log R Deviation Specification*
GDA v1.0 + ePGx	>99.0% Avg	<0.3
GSA v4.0 + ePGx	>99.0% Avg	<0.3
GCRA v1.0 + ePGx	>99.0% Avg	<0.3

*For DRAGEN Array PGx CNV analysis, Log R Dev <0.2 is required for at least 22 samples to properly normalize the baseline intensity and provide PGx CNV results. See DRAGEN Array User Guide for more details.

Evaluate performance of the PGx TGA Control Probe in GenomeStudio 2.0

1. Create the GenomeStudio 2.0 Genotyping Workspace.

• Download and install the current version of GenomeStudio, v2.0.5, from the GenomeStudio Support Resources page.

• Files required:

  • Intensity data (.idat) files from the scanned BeadChips

  • Manifest: .bpm file*

  • Cluster file: .egt file*

• *Download product files from the BeadChip Product Files page:

  • Infinium Global Diversity Array + Enhanced PGx Product Files

  • Infinium Global Screening Array v4.0 + Enhanced PGx Product Files

  • Infinium Global Clinical Research Array + Enhanced PGx Product Files

• Create the GenomeStudio workspace as described in the GenomeStudio Genotyping Module v2.0 User Guide.

• Calculate call rates and evaluate sample performance per specifications in Table 1.

1. Analyze the TGA Control Probe.

• Right-click on the Name column in the Full Data Table in GenomeStudio and select Find.

• 
• In the search window, type "TGA_Ctrl_5716" which is the name of the TGA control probe in the manifest.

• 
• Examine the SNP Graph to determine if any samples fall below a normalized R (Norm R) of 1. Select the samples with R < 1 to highlight in the SNP Graph and Samples Table for further evaluation.

  
• Evaluate the overall performance of samples with a failed TGA control. Determine if there are any other associated run failures by evaluating the data in GenomeStudio, including in the Controls Dashboard.

If samples do not meet performance expectations for R > 1, exclude them from further analysis, or flag them as having potential inaccuracies in genotype and star allele calls for the PGx content.

TGA Control Probe in DRAGEN Array

DRAGEN Array software produces genotype summary files (gt_sample_summary.csv and gt_sample_summary.json) that contain various QC metrics. The TGA_Ctrl_5716 Norm R field contains the Normalized R value of one probe where < 1 indicates the sample failed in the TGA process. This field is included in genotype summary files only for BeadChips with PGx content. For more information, refer to the DRAGEN Array User Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #5491), contact Illumina Technical Support [e-mail].