Sequencing/Genotyping
Our expertise is focused primarily on DNA sequencing, DNA fragment analysis (microsatellites, FLPs), SNP genotyping, assay design, and integrated data handling infrastructure. The GCF uses state-of-the-art robotic liquid handlers and sensitive analysis equipment for processing high throughput, low volume, automated genetics technologies.
The GCF uses ABI BigDye v3.1 dye terminator sequencing chemistry and the new generation ABI PRISM 3730xl capillary DNA analyzer for sequence analysis. The sequencing chemistry was optimized using robotic systems to miniaturize the reactions and reduce costs. For resequencing projects, we also have experience detecting SNP's in 2 deep sample pools (4 chromosomes). The 3730xl capacity exceeds 1,200 sequence runs of 600-1000 bases per day (~1 million bases per day). Sequence Analysis Version 5.1.1 is used for base calling and Sequencher Version 4.5 (both Mac and PC, GeneCodes) and Mutation Surveyor v2.5 (SoftGenetics) for multiple sequence alignments to reference sequence, variation scoring and and automated data export. Data can be exported in a number of formats (ie, an annotated Sequencher project (samples aligned to reference and SNP's scored) that can be viewed with a free, demo version of Sequencher). The GCF averages >150,000 sequence analyses per year and has a current total capacityof >450,000/year.
The GCF offers cost effective, low reaction volume, high throughput PCR that includes PCR assay design and optimization for long-range, allele specific, gene specific (pseudogene and duplication) and difficult template PCR for a variety of downstream analyses including sequence analysis, fragment length analysis, and SNP genotyping. Capacity is > 44,000 PCR reactions/day, carried out in either 96 well and 384 well formats.
The GCF offers a wide range of genotyping methodologies for candidate gene, linkage, association, and linkage disequilibrium mapping studies including re-sequencing, fluorescent microsatellite and fragment length polymorphism (indels, VNTR's, etc), SNP analyses using TaqMan allele specific 5'nuclease (ABI), AcyloprimeFP template directed dye incorporation (PE), and SNaPshot multiplex primer extension (ABI) assays, and gene duplication/deletion analyses using TaqMan qPCR. Linkage mapping projects in both human (5cM resolution ~800 markers) and mouse (~ 400 markers) are performed using the GCF's collections of 5' fluorescent labeled microsatellite markers. Custom marker panels are designed and quality tested for custom fine mapping and LOH studies. Large scale, high throughput projects are designed, processed using automated low volume liquid handlers, analyzed using a ABI PRISM 3730xl DNA analyzer, scored using GeneMapper v4.0 for rapid and accurate allele assignment and reported in either standard linkage format or in an investigator identified database format. Our current SNP genotyping methods are performed using either the Applied Biosystems (ABI) TaqMan 5' nuclease allele discrimination (ABI PRISM 7900HT Sequence Detection System; analysis capacity 250,000 genotypes/day) or the Perkin Elmer Acyloprime-FP (FP-TDI) assay platform (Perkin Elmer Envision HTS microplate reader; analysis capacity 270,000 genotypes/day). The recently acquired Illumina BeadArray Station, with a capacity to analyze 300,000 genotypes/day, expanded GCF services to include highly multiplexed SNP genotyping (GoldenGate assay), whole genome genotyping (Infinium assay), BeadArray expression analysis (Direct hyrbridization and DAZL). Both the GoldenGate SNP genotyping and Gene Expression hybridizations assays are now performed by Illumina-trained GCF staff using existing liquid handling instrumentation.
Projects using optimized PCR and DNA sequencing assays: For projects to study genomic variation in small to large sample sets, the GCF offers optimized re-sequencing screens. Using genomic sequence identified by the investigator, the GCF designs PCR primers (including informatics to identify pseudo genes, gene duplications, alternate splice variants, 5' UTR variants, etc), validates small volume PCR, optimizes sequencing assays, processes the project sample set using automated robotic liquid handling, identifies variants using Sequencher v 4.5 and Mutation Surveyor 2.5, and reports fully annotated results that includes custom annotation tracks for the UCSF genome browser. More than 40 compete gene re-sequencing projects have been performed.
The GCF has real-time and quantitative PCR capabilities using TaqMan expression probes and our ABI PRISM 7900HT Sequence Detection System.
using DHPLC, the GCF offers DHPLC heteroduplex mutation detection.
The GCF maintains and archives all data generated in the facility and can easily integrate custom data transfers as needed. The IT infrastructure includes a secure client-server network with a central LINUX Dell PowerEdge 4600 fileserver with 1.2 TB SCSI RAID 10 storage, several MAC OSX and PC client/workstations, a Windows XP Application server, an UNIX HTTP (APACHE) and FTP server, and an UNIX email (qmail) and application server (Primer3 and UNIX open source informatics tools).
In the near future, the GCF plans to implement quantitative, scalable, high density SNP genotyping. We are developing cost effective assays to develop complete and accurate genotyping solutions for specific gene variants and duplication/deletion variants (cytochrome P450s, APOE) likely to be utilized by multiple customers. We are also developing a web-based interface for clients to retrieve project timelines and finished data.
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