# Illumina

## Randomly Ordered Fiber Optic Arrays

The randomly ordered BeadArrayTM technology was invented by Professor David Walt and colleagues at Tufts University and further developed at Illumina, Inc., where it has been used as a platform for a wide range of assays (Ferguson et al., 2000; Michael et al., 1998; Walt, 2000). An array of wells is patterned into an optical imaging fiber bundle. This process takes advantage of the intrinsic structure of the optical fibers in the bundle.

The optical imaging fiber bundles used by Illumina consist of ~ 50,000 individual fibers fused together into a hexagonally packed matrix, and therefore can hold up to ~ 50,000 beads, each ~ 3 microns in diameter and spaced ~ 5 microns apart. The entire array is ~ 1.4 mm in diameter. The beads are stably associated with the wells under standard hybridization conditions. A wide variety of bead types can be used, including silica and polystyrene.

## Decoding Randomly Assembled Arrays

Since the assembly of beads into wells is a random process, the location and identity of beads in the array must be determined post-assembly. This process is called decoding. Randomly assembled arrays can be decoded by simple DNA hybridization techniques. DNA hybridization is sufficiently specific to resolve easily at least several thousand simultaneous oligonucleotide hybridizations. The approach is illustrated by the following example. Imagine an array that contains 16 types of bead, each differing by the oligonucleotide sequence attached to them.

In order to decode the array, a set of 16 “decoder” probes complementary to each of the 16 probes in the array is synthesized. Each decoder probe is then labeled with each of 4 different fluorophors to generate 4 versions of each decoder probe. Thus, for decoder 1, there are four versions 1A, 1B, 1C, and 1D, where A, B, C and D represent different labels. Individual decoders are then pooled so that the pool contains one version of each of the 16 decoders. For example:

Pool 1 = (1A, 2A, 3A, 4A, 5B, 6B, 7B, 8B, 9C, 10C, 11C, 12C, 13D, 14D, 15D, 16D)

Hybridization of Pool 1 to the array partially decodes the array, because positions that contain probes hybridizing to sequences 1, 2, 3, and 4 are illuminated by the “A” fluorophor. Positions hybridizing to sequences 5, 6, 7 and 8 are illuminated by “B”, and so on. At this stage, the identities within each group of four sequences are still ambiguous. This is resolved by a second hybridization:

Pool 2 = (1A, 2B, 3C, 4D, 5A, 6B, 7C, 8D, 9A, 10B, 11C, 12D, 13A, 14B, 15C, 16D)

The decoding process scales well and is very efficient. If two fluors are used, then 2n sequential hybridizations can distinguish 2n sequences. With 4 fluors, 4n sequences can be distinguished, and so on. The important point is that an exponential number of codes is obtained using only a small number of fluors and a small number of decoding steps. The accuracy of decoding is estimated to be 99.99%, which is more than sufficient for our needs (because each bead type is represented by at least five individual beads, the impact on assay results of an error rate of 1 in 10,000 beads decoded is negligible).