OHSU

Illumina Bead Arrays

The Illumina BeadChip is proprietary method of performing multiplex gene expression and genotyping analysis. The essential element of BeadChip technology is the attachment of oligonucleotides to silica beads. The beads are then randomly deposited into wells on a substrate (for example, a glass slide). The resultant array is decoded to determine which oligonucleotide-bead combination is in which well. The decoded arrays may be used for a number of applications, including gene expression analysis and genotyping. Scroll to the bottom of the page for a primer on array decoding.

Expression array overview

Gene expression analysis is performed using a 79-base oligonucleotide that has two segments. The 5′ 50-base segment of the oligonucleotide is designed to hybridize to sequences available in the public data repositories. It is this segment that will bind to the labeled target derived from the poly(A) component of the total RNA. The 3′ 29-base segment of the oligonucleotide is the address. The address is a unique sequence created by Illumina specifically to allow unambiguous identification of the oligonucleotide after it has been deposited on the array.

Illumina_schematic

Arrays may have as many as 44,000 unique oligonucleotides. Each oligonucleotide is synthesized in a large batch using standard technologies. The oligonucleotides are then attached to the surface of a 3-micron silica bead. Each bead has only one type of oligonucleotide attached to it, but it has hundreds of thousands of copies of this oligonucleotide.

Standard lithographic techniques are used to create a honeycomb pattern of wells on the surface of glass slides. Each well can hold one bead. The beads for a given array are mixed in equal amounts and deposited on the slide surface. The beads occupy the wells in a random distribution. Each bead is represented by, on average, about 20 instances within the array. The identity of each bead is determined by decoding using the address sequence. A unique array layout file is then associated with each array and used to decode the data during scanning of the array.

 The Illumina BeadChip is a relatively new method of performing multiplex gene analysis. The essential element of BeadChip technology is the attachment of oligonucleotides to silica beads. The beads are then randomly deposited into wells on a substrate (for example, a glass slide). The resultant array is decoded to determine which oligonucleotide-bead combination is in which well. The decoded arrays may be used for a number of applications, including gene expression analysis and genotyping. Scroll to the bottom of the page for a primer on array decoding.

Illumina Expression Array Protocol Outline

RNA is amplified using a modified Eberwine T7-based amplification protocol, using an Illumina-specific kit from Ambion. (See Figure 2).

The basics of the RNA amplification are:
  • Hybridization of a oligo-dT oligonucleotide to the polyA component of the total RNA. The oligonucleotide also has the sequence for a viral T7 RNA polymerase promoter.
  • Extend the cDNA, then synthesize a second strand to generate double stranded cDNA.
  • Add T7 RNA polymerase and nucleotides to linearly amplify the RNA. The nascent aRNA incorporates biotin-modified dUTP.
  • Hybridize the biotin-modified aRNA to the BeadChip.
  • Stain the BeadChip with Cyanine 3 derivatized to streptavidin.
  • Scan on a high resolution Illumina BeadStation scanner.
Illumina_RNA_amplification

Custom Genotyping Overview

Custom genotyping technology allows the user to integrate large numbers of samples for a select panel of defined SNPs. The maximum number of SNPs for a single screen is 1536. Sample numbers are based on 96-well plates.

Genomic DNA is normalized to 50 ng/ul and 250 total ng in 5 ul

Genomic DNA is chemically reacted to incorporate biotin. The biotin label is then used to attach the DNA to paramagnetic beads treated with streptavidin. The paramagnetic beads (not to be confused with the silica beads of the BeadChip array itself) are then used to recover DNA from solution.

Three oligonucleotides are designed for each SNP. Two are allele-specific oligonucleotides (ASO) and one is a locus-specific oligonucleotide (LSO). Each ASO has a 3′ base that is complementary to one of the two SNP bases. The LSO hybridizes downstream of the ASOs. Each oligonucleotide contains a generalized primer sequence for PCR – P1 and P2 on the ASOs and P3 on the LSO. The LSO also contains an address sequence that will be used to characterize the PCR product.

After extension and ligation, DNA is amplified using polymerase chain reaction (PCR) and labeled P1 and P2 oligonucleotides. One of these is labeled with Cy5, the other with Cy3. PCR products are separated from unincorporated material using a filter plate.

PCR product is then hybridized to a Sentrix Array Matrix (SAM, see below). Cy5- and Cy3-labeled material binds in proportion to the relative abundance of the two alleles in the sample – so that a homozygote for the allele has only one color and a heterozygote has two. Based upon the color distribution at each allele, the genotype of the samples for the designated SNPs can be determined.

Unlike the gene expression BeadChips, the genotyping beads do not use 79-base oligonucleotides. For genotyping, the only sequence attached to the bead is the 29-base address sequence. Discrimination among genotyping loci is built into the ASOs and LSOs and the SAM is used for quantitation after the loci-specific amplification.

Illumina_custom_genotyping

Sentrix Array Matrix

The Sentrix Array Matrix (SAM) is a platform for performing multiplexed genotyping. Each matrix is an array of fiber optic bundles. The bundles can be arranged in sets of 96, 384, and 1536, with the actual number available dependent upon the arrangement of laboratory thermocyclers. The current set up for OHSU uses 96-well SAMs.

The caption for the image describes the layout and use of a SAM. Each SAM has a collection of fiber optic bundles that are capped by a surface with honey comb wells designed to hold up to 1536 bead types. As with the genotyping, there is extreme redundancy in the layout of the beads and the beads are randomly distributed on the surface of the bundle..

Sentrix Array Matrix – the image shows the bottom of the SAM. Each dimple is a fiber optic bundle. Each bundle is capped with a full complement of up to 1536 oligonucleotides. In use, the SAM is inverted and the bundles are inserted into the wells of a microtiter plate – with each well containing a unique sample.

Illumna_sam_sm


Illumination of the individual sets of probes is done via the fiber optic bundle. Each bundle is capped by a single bead. Excitation light traverses the bundle and excites the fluors attached to the beads. Emitted light is then collected at the surface of the bundle.

Illumina_lightpath

Whole Genome Genotyping - Infinium II Assay

 1.  Genomic DNA is amplified using a proprietary Illumina whole genome amplification protocol.  This protocol produces faithful copies of the source DNA.  Because of the amplification step, small amounts of genomic DNA - 200 to 750 ng depending upon the array type -are sufficient for the assay.

2.  Amplified DNA is enzymatically fragmented.  End-point fragmentation is used to produce a consistent size of terminal fragments.

3.  The fragment amplified DNA is recovered and applied to the Whole Genome Genotyping Array for hybridization overnight. 

4.  The hybridized array is washed and the probe undergoes single-base extension with tagged terminating nucleotides.  A and T carry one tag; G and C carry another.  Because of this arrangement, A <-> T and G <-> C changes are not measurable.  However, this still permits the interogation of about 83% of known SNPs.

5.  The extended probe is then stained with a sandwich-staining protocol that amplifies the signal beyond what was accomplished with the whole genome amplification protocol. 

5.  The stained array is scanned on the BeadArray Reader.

 

infinium workflow 

Decoding Illumina Arrays

Once the beads have been deposited onto the glass surface, it is necessary to identify which transcript probe is in which well. This is done using the address segment of the oligonucleotide. Decoding the arrays, a process done at the Illumina facility and not part of the end user protocol, involves sequential hybridization of differentially labeled probes. The differential labeling uses three states – carboxyfluorescein (FAM) labeled green, cyanine 3 (Cy3) labeled red, and not labeled. During any given cycle of the process, a bead is green, red, or blank.

Labeled decode oligonucleotides are hybridized to the arrays at high concentrations, which allows for rapid (several minute) hybridizations, followed by washing to removed non-specific signal and background. If one assigns a number to each state – 0 to blank, 1 to green, and 2 to red, then each cycle of the process generates a trinary digit. If we look at one hypothetical probe, then the first round may be red and generates 2 for the first digit. The second round is green and generates 1, so the number is now 21. The third round red, so the number is now 212. Each round just adds a new digit to the number. This continues until there are sufficient digits to uniquely identify each probe. One digit can uniquely identify three probes (0, 1, or 2). Two digits can uniquely identify 9 probes (00, 01, 02, 10, 11, 12, 21, 22, 23). Three digits can identify 27 (we'll leave anything further as an exercise for the reader!). Rehybridization continues until there is sufficient data to unambiguously determine the identity of each bead.

A decoding example from Illumina is shown the figure. Note that the image in Section A is not the image decoded in Section C. The isolated spot in A has the code 11012202. The spot profiled in C has the code 02212110.

Illumina_decode

From Gunderson et al., Decoding Randomly Ordered DNA Arrays, Genome Research, 14, 870-877, 2004