Introduction to SNP Genotyping: Understanding Genetic Variation and Its Applications
Single-nucleotide polymorphisms or SNPs are DNA sequence variations that occur when a single nucleotide — A, T, C, or G — in the genome differs between members of a species or paired chromosomes in an individual. For example, a SNP may replace cytosine (C) with thymine (T) in a specific position in the genome. SNPs are the most common type of genetic variation event among people. No two individuals, except identical twins, have exactly same set of SNPs. SNPs help scientists map genes that cause or influence certain diseases or conditions, to identify genes important for drug response, and to understand ancestry mapping and evolution. SNPs genotyping plays a vital role in such research.
SNP Genotyping Methods
There are several different methods used for SNP genotyping and analysis but the most widely used methods are described below:
DNA Microarrays - Microarray uses a DNA chip with hundreds, thousands, or even millions of known SNP locations spotted onto it. Fluorescently labeled DNA samples are hybridized to the chip. A scanner detects the hybridized DNA at each SNP location and identifies the DNA base at that location based on its fluorescence.
TaqMan - It is a PCR-based assay that uses the 5' exonuclease activity of Taq polymerase during PCR to detect a specific SNP Genotyping and Analysis. Two short oligonucleotide probes that anneal to the DNA either side of the SNP location are used. One probe contains a reporter dye and a quencher dye. During PCR, if the probe hybridizes to the variant sequence, the reporter dye and quencher dye are cleaved and separated, resulting in detection of fluorescent signal.
Mass Spectrometry - It detects SNPs based on differences in molecular mass between SNP alleles. Following PCR amplification of the target region, primer extension is performed with labeled nucleotides. Extension products are subjected to mass spectrometry which determines the molecular weight of the final extension product to determine the SNP genotype based on the added mass from the labeled nucleotide.
DNA Sequencing - Next generation sequencing directly sequences the DNA samples and SNPs are identified from variations in the sequence reads. Sanger sequencing is used for low throughput verification of SNPs identified by other methods.
Luminex - This method uses fluorescently labeled microspheres and SNP-specific probe pairs in multiplexed format. Labeled DNA hybridizes to the microspheres and signal intensities of two reporter dyes determine which alleles are present.
SNP Genotyping Analysis
After SNP genotyping is performed, the data analysis is equally important to extract meaningful biological insights. Here are some of the key aspects of SNP genotyping data analysis:
- Quality Control - Raw genotyping data needs quality control to filter out poor quality genotypes, samples, and SNPs based on detection rates, deviation from Hardy-Weinberg equilibrium, duplications, etc. This improves accuracy of downstream analyses.
- Normalization - Different intensity signals from the different plate layout positions are normalized to remove any technical bias introduced during the genotyping processes such as dye effects, position effects, etc.
- Visualization - Cluster plots are generated for each SNP to visualize the intensity signals and manually validate the genotype clusters for each SNP.
- Association Analysis - Statistical tests such as allele/genotypes frequencies comparisons, chi-square test, Fisher's exact test are done to find significant associations between SNPs and disease/traits of interest.
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