Core Programs

Molecular Genetics Core Facility

Director: Simon Gregory, PhD

Manager: Karen Abramson, MPH

The mission of the molecular genetics core at the CHG is to provide world-class resources for cutting edge scientific research into human diseases.

The Center for Human Genetics (CHG) Molecular Genetics Core (MGC) was established in 1996 and has played a pivotal role in the generation of genotype and sequencing data for important scientific findings of Duke Research faculty and their collaborators. The MGC is a state of the art facility that offers a variety of experimental platforms capable of producing data for the genetic analysis of complex human disease. In 2006 and 2007 alone, the MGC generated over 850 million genotypes and over 200 thousand sequencing reads.

The MGC has the capability to run Applied Biosystems and Illumina platforms for DNA sequencing, Gene Expression, Copy Number Variation, and low, medium and high throughput Genotyping. Designed with flexibility in mind, the MGC can accommodate both small and large molecular genetic research projects.

Though the MGC has been established to primarily service Duke Researchers, we welcome inquiries from clients external to Duke. The MGC is pleased to offer the following services:

• Sample handling
  
• Pre- and post- genotyping QA/QC
  
• Uniplex SNP genotyping
  
• Multiplex SNP genotyping
  
• Whole genome association SNP genotyping
  
• DNA sequencing
  
• Microsatellite genotyping
  
• Gene Expression
  
• Copy number variation
  
• Data Storage
  

Due to the unique nature of each project, our services are customized to meet each researcher's needs. MGC faculty and staff are available to assist in study design to identify the optimal molecular platform needed to realize research goals. For further information please contact Karen Abramson.

Sample handling

Accurate sample handling is commensurate with the generation of premium quality molecular data. Accordingly, the MGC uses the Beckman Biomek FX liquid handling robot for automated sample handling. For experiments which are sensitive to DNA concentration, a portion of each DNA sample will be used for quantification using PicoGreen^® dsDNA Quantitation.

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Pre- and post- genotyping QA/QC

Pre- and post- genotyping QA/QC Formal QA and QC processes have been implemented to ensure delivery of data that meets MGC criteria and clients' requirements. QA activities include SOP documentation, training, and regular equipment maintenance. QC activities include the addition of MGC standards and customer sample replicates to each plate. As a genotyping project progresses, standard and replicate samples will be checked for concordant genotypes.

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Uniplex SNP genotyping

Uniplex SNP genotyping is carried out on the Applied Biosystems 7900HT Fast Real Time PCR System using both pre-developed and custom TaqMan^® SNP Genotyping Assays. Applied Biosystems has 4.5 million SNP assays available, including 3.5 million HapMap SNPs, 70,000 cSNPs and 160,000 validated assays. Custom TaqMan^® SNP Genotyping Assays allow you to create your own assays and support SNPs, insertion-deletions of up to six bases, and multiple nucleotide polymorphisms (MNP). Uniplex genotyping is best used to interrogate a small number of SNPs on a large number of samples. TaqMan^® SNP Genotyping Assays require only 3ng of sample per genotype.

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Multiplex SNP genotyping

The MGC supports two levels of custom multiplex SNP genotyping, both of which utilize Illumina's BeadArray^® technology. The system technology is well suited for medium, targeted genotyping projects and has the flexibility to allows 384 - 1584 multiplexed SNP assays using Goldengate chemistry which assays 3-micron beads placed into acid etched microwells. The multiplexed sample is hybridized overnight to the array containing the labeled beads and then read using the MGC's two Illumina BeadArray^® Readers. Clustering algorithms are then used to analyze the genotype data.

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Whole genome association SNP genotyping

For whole genome SNP association studies we use the Illumina Whole Genome Infinium^® Assay. Illumina's approach to whole-genome genotyping relies on the use of tag SNPs: loci that can serve as proxies for many other SNPs. Illumina scientists rationally select each tag SNP, choosing loci that provide the highest information content and even spacing across the genome. Because tag SNPs are used on every Infinium DNA Analysis BeadChip, researchers can achieve greater statistical power and genomic coverage using fewer SNPs and statistical tests compared to other strategies that use larger numbers of randomly chosen SNPs. With greater statistical power, you need fewer samples to detect association. Illumina offers beadchips that contain custom 7,600 assays through to standard 1 millions tag SNP chips.

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DNA sequencing

DNA sequencing at the MGC is carried out on an Applied Biosystems 48-capillary 3730 DNA Analyzer, with BigDye^® Terminator v3.1 Cycle Sequencing chemistry. The 3730 DNA Analyzer system is engineered for highly reliable, unattended operation of up to 48 hours. Automation features which minimize the need for operator intervention and decrease the risk of human error include an integrated plate stacker, internal bar code reader, and onboard polymer delivery system.

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Microsatellite genotyping

Although SNPs have, by and large, replaced microsatellites for generating linkage data, the capacity to generate microsatellite data remains important for correlating legacy data, or simply to investigate the potential impact that novel microsatellite polymorphisms have on genetic diseases. Microsatellite genotyping is carried out at the MGC on an Applied Biosystems 48-capillary 3730 DNA Analyzer using custom fluorescently labeled primers and Applied Biosystems GeneScan size standards.

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Gene Expression

The MGC Illumina BeadStation 500GX array readers can also be used for Illumina's gene expression products including whole-genome expression arrays, and both standard and customized expression arrays. Duke CHG researchers have used the Illumina expression arrays for expression profiling in a variety of tissues including tumors and cells associated with cardiovascular disease. Duke Researchers also have access to the Microarray Core facility directed by Holly Dressman PhD at the Center for Applied Genomics and Technology.

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Copy number variation

• Genotyping arrays
  The MGC uses Illumina's Infinium genotyping assays for copy number analysis. Without any changes to SNP content and assay format, Infinium DNA Analysis BeadChips support DNA copy number analysis in a wide variety of sample types. The Illumina platform permits a range of whole-genome arrays for copy number analysis, from the HumanHap300-Duo with 5.5kb marker spacing to the Human1M with 1.7kb marker spacing. The Illumina chips can also be used for paired (matched) sample analysis, where a normal/tumor pair can be run side-by-side on the same array using the HumanHap300-Duo, HumanCNV370-Duo, Human450S-Duo, and HumanHap550-Duo. All of these BeadChips target a high percentage of commonly reported copy number variants while at the same time allowing the discovery of novel CNVs.
  
• Genome tilepath arrays
  The MGC has developed whole genome tilepath microarrays for the high-throughput identification of chromosomal deletions, duplications, and aberrant epigenetic mechanisms, such as DNA methylation which affects gene regulation. The equipment used for the generation of tilepath arrays, the Genetix Qarray2 arraying robot, has the capacity to produce genomic microarrays at a density of 40,000 individual data points per microarray. The MGC uses an Axon GenePix 4100A dual laser system with high-resolution (5um) digital optics for microarray scanning, with BlueFuse and BioDiscovery's Nexus software used for data interpretation and identification of genomic rearrangements.

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Data Storage

Whole genome genotype, copy number or expression analyses have a propensity to generate massive amounts of data. The MGC will work with facility users to develop short term data storage and data transfer plans that will suit the specific aspects of each research project that is undertaken.

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