Applications Supported

Genotyping

Genotyping can be used to determine the genetic makeup of an individual in the region of interest.

Low to Mid-range SNP genotyping

Useful for candidate gene analysis or following genome wide association studies.

Sequenom iPlex
This technology allows multiplexing of assays for high quality SNP genotyping with manageable data outputs. Pools of up to 40 assays can be designed. It is also possible to run small numbers of assays for targeted SNP typing. Samples are processed in a 384-well plate format (minimum batch size for samples is 96). We perform assay design in-house which allows flexibility for customized analysis. A QC test is performed (for human samples), running the assays on a panel of CEPH DNA’s (90 family based samples plus 5 replicates) to determine how well the assays are performing. For assays with publically available information we will determine the concordance. Assays with a poor performance (giving pass rates less than 80% or significant number of false genotypes) will be noted. Subsequently the assays are run with the samples and data checked and manually edited and a final report provided to the user.

Illumina GoldenGate
Genotyping can be carried out on panels on 96 SNPs up to 3,072 validated SNPs or customized assays.

Large-scale SNP genotyping and Whole Genome genotyping

Illumina Infinium HD assay
The Illumina portfolio of Infinium HD beadchips feature the most advanced genomic content available for genotyping, Copy number variation (CNV) and Cytogenetic analysis. Illumina supply pre-validated chips to allow for whole genome genotyping with high density coverage up to 5 Million SNPs, but custom design is also available to allow targeted genotyping over a large region of interest with up to 200,000 SNPs interrogated.

Methylation Studies

Methylation studies enable the discovery of epigenetic changes across the genome or in a region of interest.

Sequenom Epityper
Allows the quantitation of methylation at individual CpG islands with up to 600bp read lengths providing accurate differential methylation information. The experiment can use pre-validated or custom assays. For all Epityper experiments, please contact us directly so that we can discuss the best option for your needs.

Illumina Infinium Methylation Assay
Provides quantitative methylation measurement at the single-CpG-site level, offering the highest resolution for understanding epigenetic changes, with the new high density array which covers over 450, 000 sites including all refseq genes, promoters, 5’ and 3’ region and miRNAs.

 

Gene Expression Studies

Sequenom QGE

For gene-expression the Quantitative (QGE) assay has been developed. This technology is high-throughput, based on robotic liquid handling of 384-well plates. This method has the advantage that it can be used to distinguish between alleles using any SNP present in the transcribed sequence and can be run as part of the routine Sequenom based applications. The consumable costs are low since the oligonucleotides and primers needed are not modified.

PCR reactions are performed where a single-stranded synthetic competitor oligonucleotide, identical to the cDNA of interest except for a 1bp change, is co-amplified with the cDNA. A series of PCR reactions is performed for each assay using varying concentrations of serially-diluted competitor. The relative amounts of competitor and test cDNA in each reaction are quantified using a primer extension assay. An extension primer is designed so that the 3' end anneals adjacent to the 1bp change and this is cyclically extended by one or two base pairs. Two populations of extended primer are created that differ in mass, depending on whether the primer annealed and extended through the cDNA amplicon or competitor amplicon. The relative amounts of the different extension products are then quantified by MALDI-TOF mass spectrometry (MassARRAY^TM system, Sequenom^TM) and the values used to create a regression curve. The concentration of competitor at which equal amounts of test and competitor cDNA are present in the reaction - the equivalence point, determines the concentration of the test cDNA in the sample. 

Sequenom Allele Specific Expression Analysis

To study differential expression due to allelic variance in the coding region of genes. The assay is based on the comparison of the ratio of alleles between 'affected' and 'non-affected' groups. In addition, a gDNA sample heterozygous for the allele is run alongside to establish there is no significant assay bias. There are 2 approaches to this type of analysis, firstly, quantitative - based on the QGE application, the number of each cDNA molecule can be determined if competitive PCR is used (where a third allele is used as a competitor and it's concentration is titrated to determine the transcript levels of each of the wild type alleles. Alternatively, the second approach is to perform 'relative' analysis (no competitor) and in this case the approach is based on 'allelotyping' run on the mass-spectrometer.The Gene Expression protocol uses a direct hybridisation assay in which gene-specific probes are used to detect labelled RNAs. Genome wide expression analyses can be performed using the available fixed content panels from Illumina. They offer multi-sample formats (6 samples per chip for mouse and 12 samples per chip for human panels).

Illumina Direct Hybridization

Total RNA is amplified and labelled with biotin during in vitro transcription. The amplified RNA is then hybridised overnight to the array. The array is then washed and stained with Cy3-Streptavidin complex which can be seen by the scanner. The arrays are made up of oligonuclieotide probes attached to beads which are randomly introduced to the chips. Each array will contain up to 30 replicates of the bead type and therefore thirty replicates of each probe. Each oligo is made up of the 50-base gene-specific probe and 29-base address sequence used to identify the randomly placed bead.

Alternatively, we also offer a sequencing based approach to study expression. For further details see the Sequencing pages.