18.02.06 SNP arrays Flashcards
What is a SNP and what are their genomic distribution?
Single nucleotide polymorphism (SNP) - a DNA sequence variation occurring commonly within a population (e.g. >1%) in which there is a single nucleotide change e.g. C to T. Approximately 50 million SNPs have been found in the human genome.
The genomic distribution of SNPs is not homogenous; SNPs occur in non-coding regions more frequently than in coding regions
How can SNP density be predicted?
SNP density can be predicted by the presence of microsatellites: AT microsatellites are potent predictors of SNP density, with long (AT)(n) repeat tracts tending to be found in regions of significantly reduced SNP density and low GC content
What are the different types of SNP in coding DNA?
- Synonymous SNPs, which do not affect the protein sequence, but still can affect its function.
- Nonsynonymous SNPs which do change the amino acid sequence of protein. The nonsynonymous SNPs are of two types: missense and nonsense.
Describe the three principles of SNP arrays.
- An array containing immobilized allele-specific oligonucleotide (ASO) probes.
- Fragmented nucleic acid sequences of target, labelled with fluorescent dyes.
- A detection system that records and interprets the hybridization signal.
What does the signal intensity in a SNP array depend on?
The amount of target DNA in the sample (to detect copy number), and
The affinity between target DNA and probe (binding will occur more efficiently between sequences with 100% of matched bases than those with 90% matched bases due to a SNP mismatch).
In general, what are SNP arrays used for?
In general, information obtained from the array data on the presence/absence of more than one allele is used to look for UPD and LOH, whilst the combined information on the number of alleles at individual SNP sites is used for determining copy number.
Describe the underlying priciples of Agilent and Illumina SNP arrays.
Both techniques require specialised equipment to measure signal intensity of the probe following hybridisation to the target sequence.
Computational algorithms are used to convert probe signal intensity into genotypes. Genotyping accuracy is quoted at over 95.5%.
Describe the Illumina SNP array.
Attached to each Illumina bead is a 50nt long sequence complementary to the sequence adjacent to the SNP sit.
The single base extension (T or G) that is complementary to the allele carried by the DNA (A or C) then binds and results in the appropriately-coloured signal (red or green).
What is the resolution of the Illumina SNP array?
Illumina latest array is the CytoSNP-850k. As name suggests, has 850,000 SNPs
Has 3262 targeted regions (inc ISCA and CCMC genes)
> 50kb backbone resolution
> 10kb resolution in ISCA regions
Describe the Agilent SNP array.
The assay consists of probes, 25 nucleotides long for both alleles.
The locations of the SNP locus varies from probe to probe
The target DNA binds to both probes regardless of teh allele it carries.
When the target sequence is complementary to all 25 bases of the probe the signal strength is strong
The presence of a SNP results in reduced affinity between the probe and the target sequence and a weaker signal
What is the resolution of the Agilent SNP array?
> Affymetrix array is the Genome-Wide Human SNP Array 6.0
> 1.8 million markers. 946,000 for CNV detection, 906,600 SNPs
Why is the term ‘absence of heterozygosity’ used in SNP array analysis?
Terminology, ‘absence of heterozygosity’ important as it does not imply any mechanism, see only AA, or BB pattern, nothing in the AB heterozygosity track indicating heterozygosity.
Why might a combined SNP and oligo approach be used?
SNPs do not occur evenly across the genome leading to arrays with uneven coverage, therefore the current “Gold Standard” for copy number detection is aCGH. To overcome the problem of lower overall resolution, combined SNP and Oligo arrays have been designed, whereby standard oligos are added to the SNP design to fill in the gaps and improve coverage.
What are the applications of SNP arrays in the postnatal setting?
Microarray testing is now the first line test for patients with developmental delay and/or dysmorphism in the majority of cytogenetic labs in the UK. With experience and a defined workflow the problems of interpretation and high signal-noise ratio can be overcome in order to get the benefits of a combined SNP/oligoarray platform.
Although SNP arrays provide LOH data, it is only relevant to report such cases with careful consideration – for example if a UPD syndrome is suspected in the clinical details (e.g. Prader-Willi syndrome). It is not usually relevant to report consanguinity (suggested by patches of LOH across the genome). Any UPD result should be confirmed by MS-MLPA or equivalent test.
Long stretches of Homozygosity may be useful to unmask potential recessive diseases in families where the clinician suspects/knows the involvement of a particular gene – further testing is needed to confirm a homozygous mutation in the suspected gene.
What are the applications of SNP arrays in the prenatal setting?
Prenatal testing by SNP array needs even more careful consideration. In-house experience (usually from postnatal microarray testing), a platform with minimal false positive calls, a fast turnaround time, a defined workflow, access to internal and external databases, extensive literature searching, and good communication with clinical geneticists are vital in order to provide a good service. A combined SNP/oligoarray platform provides extra reassurance for each call. SNP arrays do not require sex-matched controls, therefore the sex of the prenatal sample is not needed before set up.
