Overview

Infinium HTS assay

This section describes and illustrates the assay protocol. The assay requires 200ng of
original DNA sample as input.

Amplify DNA (Pre-Amp)

The DNA samples are denatured and neutralized to prepare them for amplification.
Figure 1 Denaturing and Neutralizing DNA

Incubate DNA (Post-Amp)

The denatured DNA is isothermally amplified in an overnight step. The whole-genome
amplification uniformly increases the amount of the DNA sample by several thousand-fold
without significant amplification bias.
Figure 2 Incubating DNA to Amplify

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A controlled enzymatic process fragments the amplified product. The process uses endpoint fragmentation to avoid over-fragmenting the sample.
Figure 3 Fragmenting DNA

Precipitate DNA (Post-Amp)

After an isopropanol precipitation, centrifugation at 4C collects the fragmented DNA.
Figure 4 Precipitating DNA

Illumina Infinium HTS Assay Protocol Guide

Infinium HTS assay

Fragment DNA (Post-Amp)

Overview

Resuspend DNA (Post-Amp)

The precipitated DNA is resuspended in hybridization buffer.
Figure 5 Resuspending DNA

Hybridize to BeadChip (Post-Amp)

Samples are applied to a BeadChip and separated by an IntelliHyb seal (or gasket). The
loaded BeadChip is incubated overnight in the Illumina Hybridization Oven. The
amplified and fragmented DNA samples anneal to locus-specific 50-mers during
hybridization.
Figure 6 Hybridize to BeadChip

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Unhybridized and non-specifically hybridized DNA is washed away and the BeadChip is
prepared for staining and extension.
Figure 7 Washing BeadChip

Extend and Stain (XStain) BeadChip (Post-Amp)

Single-base extension of the oligos on the BeadChip, using the captured DNA as a template,
incorporates detectable labels on the BeadChip and determines the genotype call for the
sample. XStain occurs in a capillary flow-through chamber.
Figure 8 Extending and Staining BeadChip

Illumina Infinium HTS Assay Protocol Guide

Infinium HTS assay

Wash BeadChip (Post-Amp)

Overview

Image BeadChip (Post-Amp)

The Illumina HiScan or iScan System scans the BeadChip, using a laser to excite the
fluorophore of the single-base extension product on the beads. The scanner records highresolution images of the light emitted from the fluorophores.
Figure 9 Imaging BeadChip

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Illumina Infinium BeadChips are sophisticated silicon-based array devices. An IntelliHyb

seal separates the sample sections of the slide so that you can run multiple samples
simultaneously.
Each individual sample section holds oligonucleotide probe sequences that are attached to
beads assembled into the microwells of the BeadChip substrate. Because the microwells
outnumber the distinct bead types, multiple copies of each bead type are present in the
array. This built-in redundancy improves robustness and measurement precision. The
BeadChip manufacturing process includes hybridization-based quality controls of each
array feature, allowing consistent production of high-quality, reproducible arrays.

Illumina Infinium HTS Assay Protocol Guide

Illumina Infinium BeadChips

Illumina Infinium BeadChips