SNPs analysis methods
- 1. SNPs Analysis Methods Presented By: Hadiah Bassam Al Mahdi Phd student, Molecular Genetics Gene 821 Techniques in Molecular Biology
- 2. • A difference in DNA sequence among individuals, groups, or populations. • Types of genetic polymorphisms: 1. SNPs 2. Sequence repeats 3. Insertions 4. Deletions 5. Recombination 6. Mutations What is Genetic Polymorphism ?
- 3. Alleles • Alternative form of a gene or DNA sequence at a specific chromosomal location (locus) • At each locus an individual possesses two alleles, one inherited from the father one from the mother • Genotype: a sum of these two alleles
- 4. Genotyping • The analysis of DNA-sequence variation is called Genotyping • Genotype = The genetic constitution of an individual • SNP genotyping is the determination of SNP loci on a whole-genome scale or within genomic regions of interest • SNP discovery strategies by Debbie Nickerson • Identifying SNPs by association for genotype- phenotype analysis of candidate genes by Chris Carlson
- 5. Consequences of polymorphisms Polymorphisms Drug metabolism Adverse Drug Reaction Disease Predisposition Disease Drug transport Drug Responders/ Non-responders
- 6. Single Nucleotide Polymorphisms (SNP) • Now that the human genome is (mostly) sequenced, attention turning to the evaluation of variation • Occur with a very high frequency about 1 in 1000 bases to 1 in 100 to 300 bases. ……..G G T A A C T T G …... ……..G G C A A C T T G …...
- 7. SNP Functional Categories SNPs can be categorized in a number of ways, the most common are by location and function (relative to a gene): 1. Coding Nonsynonymous • Missense, nonsense, frame shift 2. Coding Synonymous • Silent 3. Intronic • Splice site 4. mRNA utr • 5' UTR or 3' UTR 5. (Gene) locus region (5’ or 3’ to the gene) • ‘Near gene’ usually means within ~2000bp of gene 6. Genomic/extragenic (distant from any gene)
- 8. • In human beings, 99.9 percent bases are same. Remaining 0.1 percent makes a person unique. • Different attributes / characteristics / traits how a person looks, diseases he or she develops. • These variations can be: Harmless (change in phenotype) Harmful (diabetes, cancer, heart disease, Huntington's disease, and hemophilia ) Latent (variations found in coding and regulatory regions, are not harmful on their own, and the change in each gene only becomes apparent under certain conditions e.g. susceptibility to lung cancer) SNPs Effect
- 9. SNP Nomenclature The base-pair change is given in various forms: • A/C • T→G • C>T • 432G>C • T73C The HGVS nomenclature recommendations • "c" for a coding DNA sequence (like c.76A>T) • “g" for a genomic sequence (like g.476A>T) • “m" for a mitochondrial sequence (like m.8993T>C • "r" for an RNA sequence (like r.76a>u) Each SNP has an accession no. in the dbSNP (rs123465) • http://www.ncbi.nlm.nih.gov/snp
- 10. • Database of single nucleotide polymorphism (dbSNP) • Genome Wide Association Study • HapMap Project • 1000 genome Project SNP Databases and Projects
- 12. • Ideal SNP genotyping platform 1. High-throughput Capacity 2. Simple Assay Design 3. Robust 4. Affordable Price 5. Automated Genotype Calling 6. Accurate And Reliable Results • Over 100 different approaches, could identify Unknown SNPs or detect known SNPs. 1. Hybridization-based methods Such as TaqMan assay and SNPs Microarray. 2. Enzyme-based methods Such as RFLP-PCR 3. Sequencing Such as Sanger sequencing, Whole Exome Sequence and SNaPshot. SNPs Analysis Methods
- 13. RFLP-PCR • In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting separated by gel electrophoresis according to their size. • PCR-RFLP based on the digestion of PCR amplicon with appropriate restriction enzymes to produce distinct polymorphic fragments • Accuracy is low. • Cheapest. • Limited SNPs loci can detect.
- 14. • It called 5’-nuclease allelic discrimination assay. • It includes two allele-specific TaqMan MGB probes labeled with distinct fluorescent dyes and a PCR primer pair. • During PCR extension, the reporter and quencher dyes are released signal due to the exonuclease activity. • Easy to design the experiment and results showed in one lab work step (simple and quick) • It can detect only a known SNP in each reaction TaqMan SNP genotyping
- 15. • SNP microarray is the hybridization of fragmented single-stranded DNA to arrays containing hundreds of thousands of unique nucleotide probe sequences. • Each probe is designed to bind to a target Known DNA subsequence • Specialized equipment used to produce a measure of the signal intensity associated with each probe and its target after hybridization. The signal intensity depends upon the amount of target DNA in the sample, as well as the affinity between target and probe. Extensive processing and analysis of these raw intensity measures yield SNP genotype inferences1 SNPs Microarrays
- 16. SNP Array • Commercially available high-density SNP chips cover most of the human genome (around 100,000) • Custom chips allow researchers to choose desired markers. • It needs 2-3 days to get results • SNP array Platforms cover
- 17. • Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA. • In chain-termination PCR, the user mixes a low ratio of chain-terminating ddNTPs in with the normal dNTPs in the PCR reaction. ddNTPs lack the 3'-OH group required for phosphodiester bond formation; therefore, when DNA polymerase incorporates a ddNTP at random, extension ceases. • Up to 1000 b.p. • Reported and non reported polymorphism. • Accuracy and easy to analysis. Sanger Sequencing
- 18. • A primer extension-based method for genotyping known SNP positions through the automated DNA analyzer • Interrogate up to 10 SNPs from different amplicons in a single reaction. • It based on a single-base extension(SBE), that is, each probe binds to complementary DNA template in the presence of fluorescently labeled ddNTPs and DNA Polymerase . • Polymerase extends SBE probe by one base, adding one ddNTP to 3’ end. SNaPshot Assay
- 19. SNaPshot Assay • Each SBE probe was designed with a different length of poly(dNTP) tail to allow separation of SNaPshot products on the basis of size. • Hardly to design a panel (primer and probe) • Many steps required (multiplex PCR, SNapShopt cycling and Purification ) • 2-3 days to get results
- 20. WES-NGS • Sequencing of millions of small fragments of DNA (Exon) in parallel by technologies developed a few decades after the Sanger DNA sequencing. • NGS platforms produce gigabase (Gb)- sized nucleotide sequences from a single instrument run. Higher-throughput Higher analytical sensitivity and resolution of variants. Scalable to the entire genome (whole genome sequencing) or to specific parts of human genome (targeted sequencing). High Cost
- 21. Assignment NGS Simultaneous discovery and genotyping of SNPs. Other applications (hundreds of thousands of SNPs) Advantage: High throughput: Gb-sized nucleotide sequences/ run High sensitivity High resolution Disadvantage: bioinformatics skills and infrastructure (computer capacity and storage) $$ higher cost
- 22. Don’t forget to …. • Minimise the costs compared to how much information we are able to extract from the obtained data. • Choose the set of SNPs such that it is probable that the disease SNPs are included in the set. • High Sensitivity - High Specificity – Accurate. • Simple process - Easy to automate - High Throughput. • Multiplexing - Perform many assays at once - decrease costs
Editor's Notes
- #23: Aim: Minimise the costs compared to how much information we are able to extract from the obtained data – Minimise costs in the DNA-sample collection process – Choose the set of SNPs such that it is probable that the disease SNPs are included in the set – Minimise the number of samples times the number of SNPs to be genotyped – Control type1 and type 2 errors