SNPs are abundant in the genome of any organism. We are in the process of SNP development through RNA-seq. We want to validate them by taking a panel of 48 unrelated individuals. The polymorphic loci would be further used for association mapping.
I am facing a similar issue. I like Dr. McConnell's answer, but I agree - I think we need to know a bit more about your objective in order to advise you. Sometimes one can even (partially) answer a question like this via the Entrez databases or 1000 genomes browser, but we'd have to know more about exactly what you are doing.
One of the most common methods to validate new discovered SNP by sequencing is real-time PCR. Depending on the detection method that you use in the PCR (HRM, Sybr-green, hydrolysis probes or hybridization probes) you should choose your primer design program. Most companies offering the service for oligonucleotide synthesis also offer a service for an assay design. So you have the choice. Despite of this you should perform a blast search with the sequences that you obtained.
It should always be shown that the primers and probes are really specific for the potential SNP and do not detect some known SNPs or pseudogenes. It is desirable to use a second method to verify the results of the established assay. (For example one taqman probe based assay and one HRM-based assay). All data coming from NGS should be verified on a heterozygous DNA sample by Sanger sequencing. All result should be congruent; The database search as well as all experimental data. The final validation of the assay should be done be an inter- and intra-assay specific validation
Thank you all. My objective here is that, after getting the polymorphic SNPs i'll go for development of SNP chips.
I have recently validated the SNPs that have been identified from RNAseq. The easiest way for validating them would be to design PCR primers flanking the location of SNPs and then amplify the two polymorphic parents using those primers. The PCR product can then be purified and sent it to Sanger sequencing. I found this way to be more convenient compared to using CAPS markers. Hope this helps.....
Thank you very much Dr. Upendra Kumar. But one question comes to my mind that if i use natural population. Will it work?
You need to do a specific minimum no of chromosomes in order to define the frequency in one population and it may still not represent the different population. I think your sample size of 48 may not enough for validation . You may need to go through the guidelines. However they may work in the cohort that you are checking them.
We have used bulk CAPs and HRM assay design for this purpose. See our paper in BMC Genomics. We are about to update the toolset for Galaxy which you can use with vcf from variant detection to do this easily.
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