HMDS Shadow
HMDS Shadow

SNP Arrays

Bead Array Technology

Microarray technology allows for whole genome analysis of patient samples using a CHIP-based format in the investigation of gene expression and genotyping/copy number variation (CNV) analysis, using a multiplex approach.

Depending on the type of array for investigation, genomic DNA or total RNA extracted from the tissue samples are used as the starting material that will ultimately be hybridised to the probes on the array. Fluorescent markers are incorporated into the hybridised sequence during the protocol (one or 2 fluorochromes depending on the downstream application). The fluorescence is then imaged and quantified during scanning.

Illumina use a bead array format, in which multiple copies of each probe are attached to a bead type. The beads are randomly aligned onto the array, and are identified during imaging using a specific address code for each probe/bead type and unique decode files specific for each individual array.

Illumina BeadArray

Illumina BeadArray Technology uses 3-micron silica beads that randomly self assemble in microwells etched on planar silica slides.

Each bead is covered with hundreds of thousands of copies of the same specific oligonucleotide probe that act as the capture sequences in one of Illumina’s assays. These multiple copies, combined with multiple beads of each type per array, procides a high degree of redundancy. The bead identifier is used as an address label to decode the beads locations on the chip.

Illumina BeadArray bead

Illumina BeadArray technology is utilized for a broad range of DNA and RNA analysis applications, including gene expression (DASL) and genotyping/copy number variation (CNV) analysis (SNP arrays), currently in use in HMDS.

Illumina BeadArray reader

Once processed, the Chips are scanned on a BeadArray Reader (BAR or iScan)

SNP Arrays for Cytogenetic Analysis

A SNP (Single Nucleotide Polymorphism) is a position in the genome where the base-pairs that compose DNA differ between people

Single Nucleotide Polymorphism
  • SNP arrays use known DNA sequences in regions of known SNPs as the probes.
  • SNP arrays can detect mutations/polymorphisms in a gene sequence that may be constitutional (patient specific) or somatically acquired (disease specific)
  • SNP arrays can also be used to analyse copy number & Loss of Heterozygosity (LOH) - including copy-neutral Uni-parental Disomy (UPD)

CNVs (Copy Number Variations) are discrete regions of the genome where the copy number deviates from the normal (n=2)

Copy Number Variation
  • CNVs are identified using intensity information from consecutive SNPs/probes
  • SNP arrays can be used as an alternative to cytogenetic screening, with additional detection of LOH and UPD, however arrays cannot detect balanced translocations therefore other methods (PCR / FISH) must also be used (see summary below)
Direct Hybridization

Illumina’s Infinium chemistry is used for SNP array sample processing

Infinium HD Assay Step 1

Infinium HD Assay Step 2

Infinium chemistry is based on single base extension of whole genome amplified genomic DNA, followed by fragmentation.

Infinium HD Assay Step 3

Infinium HD Assay Step 4

Infinium chemistry utilises the single probe sequence (50mer) designed to hybridise immediately adjacent to the loci of interest, stopping one base before the interrogated marker.

Enzymatic single-base extension following hybridisation incorporates a labelled nucleotide to ensure marker specificity.

Infinium HD Assay Step 5

Subsequent dual-color florescent staining allows the labeled nucleotide to be detected by Illumina’s BAR or iScan imagining system, which identifies colour and signal intensity. For genotyping assays, the red and green colour signals specify each allele.

Dual colour fluorescent staining
Dual colour fluorescent staining

Homozygotes are indicated by red/red (eg SNP1) or green/green signals (eg SNP2), and heterozyotes are indicated by combined red/green signals (eg SNP3) visualised as yellow on capture. The figure above shows the diagrammatic representation of the allele ratio and the image to the left shows the scanned image.

SNPs are Dual Purpose Markers:

  • The log R ratio shows the intensity signals of the SNPs, which indicates gains or losses relative to normal (0)
  • B allele frequency gives information about the genotype as a measure of allelic ratio
  • The information is combined to provide copy number (CN) information; examples of which are shown in the figure below
Direct Hybridization