MATERI 1 GENETIKA 2019 Tugas [PDF]

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Genotyping and Linkage Mapping



Genotyping



Overview



What is genotyping ?







The analysis of DNA-sequence variation







Genotype = the genetic constitution of an individual



How much biodiversity  



1.7—2.0 million species Estimates to 10 million



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)



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



Classes of Mutation



An example of one simple question: How much variation is there?



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%



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



To scan the whole genomes… Not like this…….



but like this



Microcentrifuge Tube 384-well plates



96-well plates



Affymetrix genechip



Setting up the reactions



Not like this…….



but like this



Not like this…….



but like this



Applications enabled by HTP genotyping



Diagnostics, MAS, disease related genes, Domestication traits, bar coding, industrial protection of genotypes



Genotyped individuals



100,000



10,000



1,000



Plant and animal breeding for GWAS selected traits validation and candidate gene association Candidate region fine mapping



Genome-Wide Association Studies



100 Diagnostics Fingerprinting, Whole genome scans



10



10



100



1,000



10,000



Genotyped loci



100,000



1,000,000



High Throughput genotyping techniques Two main suppliers for GWA: ILLUMINA and AFFYMETRIX



Genotyped individuals



100,000



Genome-Wide Association Studies



10,000



iPLEX Gold



1,000



Sequenom



SNPlex, AB GenPlex



TaqMan



Invader SNaP 100 shot Pyroseq VeraCode GoldenGate



Illumina High-Density 1M-Duo chip



Illumina GoldenGate TaqMan assay Openarrays



10



Affymetrix Genome-Wide Human SNP Array 6.0 Illumina



iselect Infinium BeadChips



Affymetrix Targeted GeneChips 10



100



1,000



10,000



Genotyped loci



100,000



BeadChips 1,000,000



5 Basic Methodologies ….. 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



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 DNAsequence



Microarray (Based on Hybridization)  Purpose: multiple simultaneous measurements by hybridization of labeled probe



 DNA elements may be:   



Oligonucleotides cDNA’s Large insert genomic clones



Microarray technologies



Microarray chip 



Affymetrix 100k chip set 







Affymetrix 500k chip (SNP array 5.0) 







Entire genome with 100 000 SNPs (low density). 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)



Organization of a DNA microarray



1.28 cm



Hybridization of a labeled probe to the microarray



Detection of hybridization on microarray Light from laser



Hybridization intensities on DNA microarray following laser scanning



B



BB (0)



AB (0.5)



AA (1)



A



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



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



Overview of SNP array technology



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



Linkage Mapping



Overview



Types of Maps 



Physical Maps 







Cytogenetic Maps  







Complete or partially sequenced organisms Breakpoints in disease Direct binding of probes to chromosome



Genetic Linkage Maps 



Markers



What happens in meiosis… 



Leads to formation of haploid gametes from diploid cells







Assortment of genetic loci







Recombination or crossover



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.



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



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.



Development of The Mapping Population



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



Reasons why alleles at different loci may not assort independently: 1. Chance



2.Preferential Segregation (nonrandom segregation of homologous chromosomes) - hinted at but not shown in humans



non-



3.Linkage - the presence of loci measurably close together on the same chromosome.



Types of Linkage Analysis ƒParametric Lod-Score Hƒaseman-Elston Sib-Pair ƒAffected Sib-Pair and Affected Relative Pair ƒAffected Pedigree Member Method ƒVariance Components Method



Recombination frequency Total amount of recombinants



Ɵ = Total amount of recombinants + Total amount of non-recombinants



Parent



A



B



a



Gametes



Theta



50% non-rec and 50% rec



0.5



90% non-rec and 10% rec



0.1



99% non-rec and 1% rec



0.01



100% non-rec



0



b



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







 



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



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.



• 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



Lod scores



1cM = 1MB 1MB=1000kb 1kb=1000bp 1cM = 1,000,000 bp



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



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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



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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



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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



Genetic distance Genetic distance = the genetic length over which one crossover occurs in 1% of meiosis. This distance is expressed in cMorgan. 1 cMorgan = 0.01 recombinants = average of 1Mb (physical distance) (Assuming that the recombination frequency is uniform along the chromosomes)



As double recombinants occur the further two loci are, the frequency of recombination does not increase proportionately.



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).



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)



Thank You Any Questions ??