The Southern California Genotyping Consortium (SCGC)
Currently occupies approximately 750 sq/ft of dedicated laboratory space in the Gonda research facility on the UCLA campus. Installed capital equipment includes one Illumina LIMS enabled iScan reader with Autoloader II support upgraded to accommodate GoldenGate high throughput genotyping, Infinium whole genome genotyping and methylation chips as well as human, mouse and rat gene expression chips, two Tecan Genesis 150 robotic liquid handling platforms with Illumina GTS robot control software installed, one Tecan Evo 100 robotic liquid handling platform. One Tomtec autosealer, one MJ Research tetrad 2 thermocycling system and one Illumina genotyping LIMS and dedicated servers. Additional equipment includes six programmable incubation ovens, six microplate heat blocks, two tabletop Jouan centrifuges, two Molecular Dynamics fluorescent microplate readers, one speedvac and four high capacity microplate shakers.
The SCGC is a not for profit academic resource available to researchers in the Southern California region and was created to provide snp genotyping services at the lowest possible cost. Services include: project and SNP pool design assistance, sample quantitation and normalization, whole genome amplification of samples (when necessary), high throughput snp genotyping, gene expression services as well as whole genome methylation analysis.
The system is based on Illumina’s unique technology of self assembled arrays of addressed beads. Very dense arrays are created by allowing pools of beads, derivitized with unique oligo sequences to self-assemble into arrays of various densities and configurations. The arrays, with regularly spaced but randomly ordered beads, are then probed to decode the identity of each bead at each position. A decoded array is then ready to decompile a multiplexed genotyping reaction.
DNA prep for GoldenGate genotyping reactions is straightforward. Between 500 ng and 1 ug of genomic DNA is provided in 96 well plate format (96 well skirted v-bottom polypropylene microplates Cat# MSP-9601 from Biorad sealed with strip caps). Genomic DNA is quantified using a picogreen assay and normalized to 50 ng per ul. 250 ng of gDNA is used per reaction with up to 1536 snps typed in each reaction.
The multiplexed genotyping reactions themselves are based on well characterized biochemical reactions but carried out under highly controlled and optimized conditions. Oligo pools containing allele specific and locus specific oligos tailed with IllumiCode sequences and/ or universal primer sequences are created for up to 1536 unique SNP loci per pool. The oligo pools are allowed to hybridize to genomic DNA, undergo primer extension and ligation such that each allele generates a unique PCR template with common primer sequences. A multiplexed PCR generates labeled products from the templates and the amplified products are hybridized to the decoded array so that the genotype information for each locus can be scanned, decompiled and recorded.
With arrays bundled into 96 well formats and the capacity to type 1536 unique loci per reaction, the system is capable generating 147456 SNP genotypes per 96 well sample plate. The ability to process 3 plates in a normal 8 hour day gives the system the capacity to generate nearly half a million genotypes per day.
The entire process, highly automated, is controlled by a LIMS capable of tracking and organizing the workflow both at the process and project level. The LIMS integration is such that under normal circumstances it is extremely difficult to process a reaction incorrectly.
Additional quality control is facilitated by numerous built in and custom QC metrics. Each biochemical step in the protocol is monitored using dedicated bead types included in every array bundle and evaluation of qc parameters is facilitated by both the GenCall and BeadStudio analysis packages. Robot performance is monitored on a regular basis using fluorometric pipetting assays. Incubator, refrigerator, freezer and ambient room temperatures are monitored using data loggers. Pipettes are routinely calibrated and plate shaker rpm’s are calibrated using hand held tachometers.
In addition to custom GoldenGate genotyping, the SCGC supports all current Infinium whole genome genotyping chips incuding Methylation assays and all gene expression chips formats.
The SCGC is directed by Nelson Freimer (UCLA) and its management involves an oversight committee including Tony Long (UC Irvine) and Magnus Nordborg (USC). The SCGC is committed to providing equivalent access to its members at all institutions. Additionally, the SCGC will sponsor monthly scientific meetings of the user group to discuss both theoretical and practical aspects of SNP genotyping. These meetings will rotate between the campuses of UCLA, UCI, and USC (main campus).