What is the genotyping facility?

The facility based at the Institute of Child Health includes a high throughput Pyrosequencer and Affymetrix SNP arrays and associated robotics for SNP typing.

This facility is part of the genotyping core of London IDEAS, aimed at ‘capacity building' for genetics research, which also includes capillary and slab gel automated sequencers on the UCL and St. George's Hospital sites.

Kerra Pearce manages the ICH facility. Currently, access to the Pyrosequencer is available to researchers who have already been trained on the equipment. We have Affymetrix 10Kv2.0 and 50/100K mapping array systems for whole genome SNP typing projects but due to volume of work new contracts are no longer being taken on.  

Acknowledgement of major funders

The Institute of Child Health together with fundraising initiatives within the Great Ormond Street Hosptial, and Jeans for Genes, provided monies to establish the Pyrosequencer and robotics. Management of the facility is funded directly by London IDEAS.

Who can use the service?

We are contracted to collaborators to genotype cohorts for specific SNPs as part of fully funded projects across the London IDEAS consortium. Unfortunately due to volume of work we can no longer take on new projects, except for access to the Pyrosequencer for researchers who have already been trained on the equipment.

Projects completed and current

A full list of projects using the London IDEAS SNP genotyping facility is available here (Word 36k).

Pyrosequencing and SNP arrays– the technology

Pyrosequencing has a novel ‘sequencing-by-synthesis' approach. The chemistry relies on addition of dNTPs to a single stranded PCR amplified DNA template, by DNA polymerase. This releases pyrophosphate that is used in a reaction to produce ATP, which subsequently drives the conversion of luciferin to oxyluciferin, by luciferase. This generates visible light, which is detected by a CCD camera. The amount of light generated is directly proportional to the amount of ATP and therefore to the number of nucleotides incorporated.

The Pyrosequencer itself (PSQHS96A) is an off-the-shelf machine with a robotic arm capable of loading 96-well plates.

The Affymetrix Genechip uses allele specific hybridisation to genotype more than 10,000 SNPs per target DNA sample. This is an excellent technology for carrying out whole genome screening for linkage analysis, autozygosity screening, and for Loss of Heterozygosity mapping. Key individuals could be targeted, allowing more focussed work using STRPs to validate candidate linkage regions. We can run the 10K and 50/100K mapping chips.

High quality target DNA is restriction digested then amplified by PCR using a universal primer. After labelling, the PCR products are hybridised to the chip, stained with fluorescent antibody, and read in the Affymetrix Genechip scanner. Probe intensities are converted to genotypes by the Genechip Data Analysis Software, and presented as an Excel spreadsheet.