Genotyping, linkage mapping and binary data
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1) Genotyping is the analysis of DNA sequence variation to determine an individual's genetic constitution or genotype. It involves identifying polymorphisms like single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). 2) High throughput genotyping techniques like microarrays allow for the simultaneous genotyping of hundreds of thousands to over a million loci across many individuals. This has applications in disease association studies, plant and animal breeding, and forensic analysis. 3) Linkage mapping involves analyzing co-inheritance patterns of genetic markers and traits in families to determine if genes are linked (located close together) on the same chromosome. It is used to construct genetic maps of chromosome regions and identify quantitative trait loci (QTLs).
Genotyping, linkage mapping and binary data
- 1. Genotyping, Linkage Mapping and Binary Data Mohamed Atia Omar Ph.D Genome Mapping Research Dept. – AGERI – ARC FAO Training , 2014 , Egypt
- 3. What is genotyping ? The analysis of DNA-sequence variation Genotype = the genetic constitution of an individual
- 4. 1.7—2.0 million species Estimates to 10 million How much biodiversity
- 6. Important Terms Variation : Any nucleotide change in the genome Rare Polymorphism: Variation found in < 1% of population Polymorphism : Variation found in ≥1% of population Locus: Chromosomal location of a gene Allele : alternative form of a gene or DNA sequence at a specific chromosomal location (locus) Heterozygous: Feature of interest is different in both alleles Homozygous : Feature of interest is identical in each allele Hemizygous : Only one allele exists (X in Males)
- 7. What are the Types of Mutations / Polymorphisms to be Genotyped? There are six major classes of genetic variation: 1. Single base changes 2. Simple di-, tri-, tetranucleotide repeats 3. Small insertions or deletions 4. Larger, tandem repeats 5. Multi-gene (Megabase) duplication (CNV) 6. Complex rearrangements
- 9. An example of one simple question: How much variation is there?
- 12. What are the most Informative Classes for Genotyping Studies ? Polymorphism Type Nickname Heterozygosity 1. Single base changes SNP 1-50% 2. Simple di-, tri-, tetranucleotide repeats STR- short tandem repeats 50-90% 3. Small insertions or deletions INDELS - Insertions or deletion 1-50% 4. Larger, tandem repeats VNTR- variable # of tandem repeat 50-90% 5. Multi-gene (Megabase) duplication CNV - Copy Number Variation 1-50% 6. Complex rearrangements ----------- 1-50%
- 13. How many loci should be assayed? Two strategies for selecting are possible: • Select a few highly informative markers • Select numerous, poorly informative, markers randomly distributed within the genome
- 14. To scan the whole genomes… Not like this……. but like this Microcentrifuge Tube 96-well plates 384-well plates Affymetrix genechip
- 15. Not like this……. but like this Setting up the reactions
- 16. Not like this……. but like this
- 17. Genotyped loci 10 10 100 100 1,000 1,000 10,000 10,000 100,000 100,000 Genotypedindividuals 1,000,000 GWAS validation and candidate gene association Genome-Wide Association Studies Plant and animal breeding for selected traits Candidate region fine mapping Fingerprinting, Whole genome scans Diagnostics Applications enabled by HTP genotyping Diagnostics, MAS, disease related genes, Domestication traits, bar coding, industrial protection of genotypes
- 18. High Throughput genotyping techniques Genotyped loci 10 10 100 100 1,000 1,000 10,000 10,000 100,000 100,000 Genotypedindividuals 1,000,000 GoldenGate assay Infinium BeadChips iselect VeraCode GoldenGate SNPlex, GenPlex TaqMan Openarrays iPLEX Gold Pyroseq SNaP shot Invader TaqMan BeadChips Illumina AB Sequenom Targeted GeneChips Affymetrix Illumina High-Density 1M-Duo chipIllumina Affymetrix Genome-Wide Human SNP Array 6.0 Genome-Wide Association Studies Two main suppliers for GWA: ILLUMINA and AFFYMETRIX
- 21. 1) Hybridization – Microarrays – TaqMan, Molecular Beacons 2) Allele-specific PCR – FRET – Intercalating Dyes 3) Primer Extension – MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time-of-flight mass spectrometry) – SNaPshot (Single nucleotide primer extension) 4) Ligation – Padlock Probes – Rolling Circle Amplification 5) Endonuclease Cleavage – RFLP – PIRA/RFL 5 Basic Methodologies …..
- 23. RFLPs (Based on Endonuclease Cleavage) Differences in DNA sequence generate different recognition sequences and DNA cleavage sites for specific restriction enzymes Two different genes will produce different fragment patterns when cut with the same restriction enzyme due to differences in DNA sequence
- 24. Microarray (Based on Hybridization) Purpose: multiple simultaneous measurements by hybridization of labeled probe DNA elements may be: Oligonucleotides cDNA’s Large insert genomic clones
- 25. Microarray technologies DNA microarrays Ordered arrangement of multiple sets of DNA on solid support
- 26. Microarray chip Affymetrix 100k chip set Entire genome with 100 000 SNPs (low density). Affymetrix 500k chip (SNP array 5.0) Entire genome with 500 000 SNPs (high density) Affymetrix 1M chip (SNP array 6.0) Entire genome with 1 000 000 SNPs (very high density)
- 27. Organization of a DNA microarray 1.28 cm
- 28. Hybridization of a labeled probe to the microarray
- 29. Detection of hybridization on microarray Light from laser
- 30. Hybridization intensities on DNA microarray following laser scanning
- 32. SNPs Single Nucleotide Polymorphisms Change one nucleotide Insert Delete Replace it with a different nucleotide Many have no phenotypic effect Some can disrupt or affect gene function
- 33. SNP genotyping methods over 100 different approaches Ideal SNP genotyping platform: high-throughput capacity simple assay design robust affordable price automated genotype calling accurate and reliable results
- 34. Overview of SNP array technology
- 35. A little more on SNPs Most SNPs have only two alleles Easy to automate their scoring Becoming extremely popular Typing Methods Sequencing Restriction Site Hybridization
- 37. Types of Maps Physical Maps Complete or partially sequenced organisms Cytogenetic Maps Breakpoints in disease Direct binding of probes to chromosome Genetic Linkage Maps Markers
- 38. What happens in meiosis… Leads to formation of haploid gametes from diploid cells Assortment of genetic loci Recombination or crossover
- 39. What is Linkage? Linkage is defined genetically: the failure of two genes to assort independently. Linkage occurs when two genes are close to each other on the same chromosome. However, two genes on the same chromosome are called syntenic. Linked genes are syntenic, but syntenic genes are not always linked. Genes far apart on the same chromosome assort independently: they are not linked. Linkage is based on the frequency of crossing over between the two genes. Crossing over occurs in prophase of meiosis 1, where homologous chromosomes break at identical locations and rejoin with each other.
- 40. Applications/Uses of Linkage Maps Studying genome structure, organization and evolution. Estimation of gene effects of important agronomic traits. Tagging genes of interest to facilitate marker assisted selection (MAS) programs. Map based cloning Identify genes responsible for traits. Plants or Animals Disease resistance Meat or Milk Production, …… etc
- 41. Genetic Linkage Mapping Steps Development of The Mapping Population Genotyping of Mapping Population (Selection of suitable MM). Linkage Analysis Map Construction QTL Identification (in case QTL-Mapping) Marker-Assisted Selection.
- 42. Development of The Mapping Population
- 45. Linkage analysis Linkage : alleles from two loci segregate together in a family. Recombination fraction (θ): the probability of a marker and a susceptibility locus segregating independently (recombination). θ= 0.5 No linkage; θ< 0.5 linked together
- 46. 1. Chance 2. Preferential Segregation (nonrandom segregation of non- homologous chromosomes) - hinted at but not shown in humans 3. Linkage - the presence of loci measurably close together on the same chromosome. Reasons why alleles at different loci may not assort independently:
- 47. ƒParametric Lod-Score ƒHaseman-Elston Sib-Pair ƒAffected Sib-Pair and Affected Relative Pair ƒAffected Pedigree Member Method ƒVariance Components Method Types of Linkage Analysis
- 48. Recombination frequency Ɵ = A B a b 50% non-rec and 50% rec Total amount of recombinants Total amount of recombinants + Total amount of non-recombinants Theta 100% non-rec 0 0.5 GametesParent 90% non-rec and 10% rec 99% non-rec and 1% rec 0.1 0.01
- 50. In double heterozyote: Cis configuration = mutant alleles of both genes are on the same chromosome = ab/AB Trans configuration = mutant alleles are on different homologues of the same chromosome = Ab/aB
- 51. Genes with recombination frequencies less than 50 percent are on the same chromosome = linked) Linkage group = all known genes on a chromosome Two genes that undergo independent assortment have recombination frequency of 50 percent and are located on nonhomologous chromosomes or far apart on the same chromosome = unlinked
- 52. Recombination Recombination between linked genes occurs at the same frequency whether alleles are in cis or trans configuration Recombination frequency is specific for a particular pair of genes Recombination frequency increases with increasing distances between genes No matter how far apart two genes may be, the maximum frequency of recombination between any two genes is 50 percent.
- 53. • Cross-over frequencies can be converted into map units. • Ex: A 5% cross-over frequency equals 5 map units. – gene A and gene B cross over 6.0 percent of the time – gene B and gene C cross over 12.5 percent of the time – gene A and gene C cross over 18.5 percent of the time
- 54. Lod scores 1cM = 1MB 1MB=1000kb 1kb=1000bp 1cM = 1,000,000 bp
- 56. 58 Genetic Mapping The map distance (cM) between two genes equals one half the average number of crossovers in that region per meiotic cell The recombination frequency between two genes indicates how much recombination is actually observed in a particular experiment; it is a measure of recombination Over an interval so short that multiple crossovers are precluded (~ 10 percent recombination or less), the map distance equals the recombination frequency because all crossovers result in recombinant gametes. Genetic map = linkage map = chromosome map
- 57. 59 Gene Mapping: Crossing Over Crossovers which occur outside the region between two genes will not alter their arrangement The result of double crossovers between two genes is indistinguishable from independent assortment of the genes Crossovers involving three pairs of alleles specify gene order = linear sequence of genes
- 58. 60 Genetic vs. Physical Distance Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosome Recombination “hot spots” overestimate physical length Low rates in heterochromatin and centromeres underestimate actual physical length
- 59. Gene Mapping Mapping function: the relation between genetic map distance and the frequency of recombination Chromosome interference: crossovers in one region decrease the probability of a second crossover close by Coefficient of coincidence = observed number of double recombinants divided by the expected number Interference = 1-Coefficient of coincidence
- 60. Genetic distance Genetic distance = 1 cMorgan = 0.01 recombinants = average of 1Mb (physical distance) the genetic length over which one crossover occurs in 1% of meiosis. This distance is expressed in cMorgan. As double recombinants occur the further two loci are, the frequency of recombination does not increase proportionately. (Assuming that the recombination frequency is uniform along the chromosomes)
- 61. Linkage related Concepts Interference - A crossover in one region usually decreases the probability of a crossover in an adjacent region. CentiMorgan (cM) - 1 cM is the distance between genes for which the recombination frequency is 1%. Lod Score - a method to calculate linkage distances (to determine the distance between genes).
- 62. Linkage vs. Association Linkage analyses look for relationship between a marker and disease within a family (could be different marker in each family) Association analyses look for relationship between a marker and disease between families (must be same marker in all families)
- 64. Binary Data definition Binary data is data whose unit can take on only two possible states, traditionally termed 0 and +1 in accordance with the binary numeral system and Boolean algebra.
- 65. Levels of Binary Data Storage
- 76. Thank You Any Questions ??