Single nucleotide polymorphism (SNP) development and typing
Semi-automated medium-scale SNP genotyping is supported at NBAF-S using the Illumina GoldenGate Assay on a newly-acquired BeadXpress Reader System. This powerful assay allows us to perform genotyping of non-model systems by genotyping 48, 96, 144, 192 or a maximum of 384 SNPs per individual in a single reaction. While it is possible to analyse small numbers of SNPs, SNP assays are manufactured in the above multiples and it may not be economically sensible to use fewer markers than can be supplied in each price band.
Various levels of support can be provided:
– SNP discovery: usually using new-generation sequencing provided by NBAF-L (Roche 454) or NBAF-E (Illumina Solexa)
– DNA preparation and quantification
– SNP selection and array design
– SNP typing on the BeadXpress
– SNP data analysis
Numbers of SNPs
The BeadXpress is intended for 'small' SNP projects. That is, assays of 48, 96, 144, 192 or 384 SNPs.
Illumina recommend running Goldengate on an iScan for projects above 384 SNPs, a different platform (e.g BeadStation) when typing 1536, and using Infinium chips for projects above 3000 SNPs.
Normally we only offer projects up to a maximum of 384 SNPs, because we do not have the hardware to do anything larger-scale. Following discussion we could potentially run projects with 768 SNPs (by running two lots of 384 SNPs) although please note this would double the staff work load and time involved.
Numbers of samples
Within the BeadXpress framework, one has to order kits which will genotype 480 individuals. i.e. 1 kit = 480, 2 kits = 960, 3 kits = 1440 etc.
The largest number of individuals we can support is a maximum of 1440 individuals (or possibly more following discussion).
Please bear in mind that one plate of 96 samples is approximately 3 days full-time work.
(The approximate labour divisions for 5 plates: at least 1 day prep, 7 days to process and 5 days to analyse.)
Therefore 1440 individuals (15 plates) would take 3x15=45 working days = approximately 9 weeks full-time work.
The smallest project we can offer is 480 individuals x 48 SNPs.
The largest project we normally offer is 1440 individuals x 384 SNPs.
SNP development sample requirements: At least 5 micrograms of (pooled) high-quality cDNA is required from which the SNPs would be initially identified by sequencing.
For the SNP genotyping of each individual, 250 ng of genomic DNA at a concentration of 50 ng/ul (quantified using spectrophometer/fluorometer). DNA should be buffered in 10 mM Tris-HCl pH 8.0, 1mM EDTA.
Individuals are genotyped in multiples of 480, so experiments should be designed with this in mind.