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High-Throughput Polymorphism Detection Core

The High-Throughput Polymorphism Detection Core (HTPC) provides services to DF/HCC investigators conducting molecular analyses of germline DNA collected as part of a wide range of investigations into the molecular epidemiology of cancer, including Genome Wide Association Studies (GWAS). This Core provides high-throughput assays of known gene mutations and polymorphisms (SNPs),  and flexible, high quality, high-throughput SNP Genotyping to the DF/HCC research community.

The Core has two sites where genotyping and other services are performed: 1) HSPH, under the supervision of Immaculata De VivoBWH; and 2) BWH-Cambridge, under the supervision of David KwiatkowskiBWH.

The human genome project and the advent of relatively inexpensive deep sequencing have led to an explosion of information regarding human genetic variation, including both SNPs and copy number changes. In turn, these advances have led to a large number of GWAS studies, which have provided detailed information on alleles associated with human disease. This has resulted in a muchhigher demand for genotyping information from epidemiologists. Understanding the genetic contribution to cancer is moving from the search for high penetrance genes (such as BRCA1 and BRCA2) to medium- and low-penetrance alleles more common in populations. Clinical investigators are also studying genetic variants that may alter response to therapy and determine prognosis; these variants are likely to be common alleles of modest effect, as in drug metabolism genes. Most of the common variation on the human genome is accounted for by SNPs.

DF/HCC has a large community of population science and clinical researchers who are conducting case-control and cohort studies of a wide variety of cancers. DF/HCC members need to be able to genotype substantial numbers of SNPs in their studies. The Core enables such studies at a reasonable cost and the highest quality. 

The principal focus of the Core is high-throughput genotyping. The platforms used are Applied Biosystems TaqMan, OpenArray, Sequenom and Illumina. In addition to the singleplex TaqMan genotyping platforms, the Core also has a TaqMan Open Array SNP genotyping platform (OpenArray). This platform allows users to run 16, 32, or 64 SNPs on one OpenArray. The Illumina GoldenGate Assay allows custom panels of SNPs that are available in 96 or 384-1,536 assays per OPA (Oligo Pool All) tubes. Sequenom allows runs of 40 multiplexed panels of SNPs in a single well of a 384-well plate using Sequenom iPLEX chemistry. The advantage of a diverse range of technologies is that the Core is able to tailor services to member needs and research objectives. The Core also provides high-volume DNA extraction and sample handling. There is substantial capacity in DNA extraction, quantitation, and mother plate preparation. 

Key services provided by the High-Throughput Polymorphism Core include: 

Applied Biosystems TaqMan SNP. Allelic discrimination is performed on the ABI7900. In this method, specialized probe oligonucleotides are used to distinguish the SNPs during or at the end of a PCR reaction.

Applied Biosystems OpenArray. Genotyping is a nanofluidic chip based platform that allows the Core to run assays that are known to work well using traditional 384- well format TaqMan assays. The OpenArray system allows users to run 16, 32 or 64 SNPs on one OpenArray. Depending on the number of SNPs on an OpenArray up to 144 samples can be run per OpenArray (in a 16-plex assay).

Sequenom MassArray. This is suitable for small numbers of SNPs, from two to 300 SNPs. It is performed using MALDI-TOF (Matrix-assisted laser desorption ionization - time of flight) mass spectrometry. The service includes primer and assay design through to production of genotypes. All steps involved are highly automated and are tracked using a laboratory management system with bar coding. An average of 34 SNPs can be plexed into a single well of a 384 well plate using Sequenom iPLEX chemistry, with a maximum of 40 SNPs per plex. Specialized equipment for this work includes a Biomek multi-pipettor robot, a Multimek multi-pipettor robot, a nanoliter plotting robot for spotting the extension products onto chips and the mass spectrometer.

Illumina Bead Station GoldenGate. Assay system for SNP genotyping is most suitable for large-scale association studies, such as whole genome linkage mapping panels and large-scale fine mapping panels. The genotyping is carried out in 384 plex reactions, then in multiples of 96 SNPs up to 1,536 SNPs using Illumina custom SNP panels. Many standard panels are available from Illumina.

Illumina Bead Station Infinium. This assay system is used for Whole Genome Genotyping. Genotyping is carried out on standard chips containing up to one million SNPs. Custom genotyping of large numbers of SNPs is available using the iSelect system. Several standard chips allow the addition of iSelect custom SNP sets to allow customization of the chip for the userís needs.

Quantitative PCR (qPCR)-based Relative Telomere Length (RTL) Assay. RTL of peripheral blood leukocyte DNA will be assayed using the qPCR-based method modified for a high-throughput 384-well format. Telomere length measured in surrogate tissues, such as peripheral blood leukocytes and buccal cells, has emerged as a putative biomarker of chronic disease risk. The core uses this method to assess relative telomere length of high quality DNA derived from either buffy coat or buccal cells.