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Understanding Genotyping
Genotyping is the process of determining the DNA sequence—the genotype—at specific positions within a gene of an individual. Sequence variations can be used as markers in linkage and association studies to determine genes relevant to specific traits or disease.
Genotyping is used to identify variation at specific positions in the DNA sequence of any biological species, from microorganisms to humans. This variation, the genotype, occurs naturally and can be considered a genetic fingerprint of an individual. It is identified as distinct when compared to a reference sequence derived from the general population or a defined subgroup, and can differ from the reference sequence in numerous ways. Types of variation include single base changes (commonly referred to as single nucleotide variants and polymorphisms, or SNVs and SNPs), insertions and deletions (indels), and even the number of gene copies (copy number variation, or CNV).
SNPs are the most common type of sequence variant; they are typically defined as SNVs that occur at >1% in the population. Based on the number of SNPs cataloged in Build 149 of the SNP database, dbSNP, maintained by the National Center for Biotechnology Information (NCBI) [1] and a genome size of 3.4 x 109 bp [2], the human genome should contain a SNP approximately once every 22 bases! Other common model systems show a similarly high frequency of SNPs [3].
A series of adjacent SNPs from an individual represent a haplotype and can serve as a signature for a specific phenotypic trait, such as fruit production or certain cancers (Figure 1).
By comparing sequence variations among individual plants, animals, or humans, researchers can identify heritable genes relevant to specific traits. These unique differences can be used as markers in linkage and association studies to:
- Map gene function and genetic variation
- Identify gene variants associated with a unique phenotype or disease; where SNPs serve as genetic markers (biomarkers)
- Match effective medical treatments to populations with specific genotypes (personalized medicine)
- Facilitate animal breeding, such as the selection of a desired genotype from a cross hybrid
- Trace ancestry or the origins of disease; map evolution, such as phylogenetic relationships between species; or test for family relationship for inheritance or paternity
- Carry out population association studies, such as genome-wide linkage analysis, which use SNPs as markers
- Perform forensics, using genotyping to identify a specific individual
- Do pathogen typing and resistance screening
- Monitor biodiversity
As mentioned above, there are also other types of information collected apart from sequence differences, such as copy number variation (CNV).
Technologies used to study genotypingThere are different approaches to SNP genotyping with the number of samples, the number of genotypes to be tested, and the amount of sample material available, all factoring into choice of technology. Methods include whole genome analysis by NGS or microarrays, or more targeted analysis using qPCR, dPCR, or targeted sequencing.
Experimental design can be low throughput vs. highly multiplexed (look at numerous SNPs simultaneously). Common analysis methods include end-point or quantitative PCR (qPCR), targeted sequencing, bead or microarray analysis, and even mass spectrometry.