Self-assembled random arrays: High-performance imaging and genomics applications on a high-density microarray platform

Illumina is developing a BeadArray(TM) technology that supports SNP genotyping, mRNA expression analysis and protein expression analysis on the same platform. We use fiber-optic bundles with a density of approximately 40,000 fibers/mm(2). At the end of each fiber, a derivatized silica bead forms an array element for reading out a genotyping or expression assay data point. Each bead contains oligonucleotide probes that hybridize with high specificity to complementary sequences in a complex nucleic acid mixture. We derivatize the beads in bulk, pool them to form a quality-controlled source of microarray elements, and allow them to assemble spontaneously into pits etched into the end of each optical fiber in the bundle. We load our fiber bundles, containing 49,777 fibers, with up to 1520 different bead types. The presence of many beads of each type greatly improves the accuracy of each assay. As the final step in our manufacturing process, we decode the identity of each bead by a series of rapid hybridizations with fluorescent oligos. Decoding accuracy and the number of beads of each type is recorded for each array. Decoding also serves as a quality control procedure for the performance of each element in the array. To facilitate high-throughput analysis of many samples, the fiber bundles are arranged in an array matrix (Sentrix(TM) arrays). Using a 96-bundle array matrix, up to 1520 assays can be performed on each of 96 samples simultaneously for a total of 145,920 assays. Using a 384-bundle array matrix, up to 583,680 assays can be performed simultaneously. The BeadArray platform is the highest density microarray in commercial use, requiring development of a high-performance array scanner. To meet this need, we developed the Sherlock(TM) system, a laser-scanning confocal imaging system that automatically scans all 96 bundles of an array matrix at variable resolution down to 0.8 Micron. The system scans with both 532 and 635 nm lasers simultaneously, collecting two fluorescence images. The optical train is designed around a telecentric, flat field, macro scan lens with a field of view of 2 nun. Our BeadArray platform is adaptable to many different assays. In our genotyping services lab, we automated the development and production of highly multiplexed SNP genotyping assays. Each SNP call is made automatically and assigned a quality score based on objective measures of allele clustering across multiple samples. The quality score correlates directly with genotyping accuracy. With a small number of robots and thermal cyclers, and a team of 5 people, we have the capacity to perform over I million genotypes per day. The system is modular so that scale-up is limited only by demand. The system has the capacity, versatility, and cost structure to meet the needs of large-scale genomic analysis.