Two technologic breakthroughs have enabled the application of SNPs to high-throughput “gene hunting.” First is the completion of several large genomic sequencing projects (the HapMap project and the 1000 genome project) that have provided linkage disequilibrium patterns in multiple ethnoracial groups, based on genomewide SNP mapping. The entire human genome can now be divided into blocks known as “haplotypes,” which contain varying numbers of contiguous SNPs on the same chromosome that are in linkage disequilibrium and hence inherited together as a cluster. As a result, rather than querying every single SNP in the human genome, comparable information about shared DNA can be obtained simply by http://ebooksmedicine.net
A AC C C G T GG A B T A AC C GG T Add all four nucleotides, labeled with different dyes C G T Wash, four-color imaging C G T C G T C G T G A C G A C Cleave dye and terminating Repeat C A T G groups, wash
Top: CATCGT Bottom: CCCCCC
Fig. 7.41 Principleofnext-generationsequencing.Severalalternativeapproachescurrentlyareavailablefor“NextGen”sequencing,andoneofthemorecommonlyusedplatformsisillustrated.(A)ShortfragmentsofgenomicDNA(“template”)between100and500basepairsinlengthareimmobilizedonasolidphaseplatformsuchasaglassslide,usinguniversalcaptureprimersthatarecomplementarytoadaptersthathavepreviouslybeenaddedtoendsofthetemplatefragments.Theadditionoffluorescentlylabeledcomplementarynucleotides,onepertemplateDNApercycle,occursina“massivelyparallel”fashion,atmillionsoftemplatesimmobilizedonthesolidphaseatthesametime.Afour-colorimagingcameracapturesthefluorescenceemanatingfromeachtemplatelocation(correspondingtothespecificincorporatednucleotide),followingwhichthefluorescentdyeiscleavedandwashedaway,andtheentirecycleisrepeated.(B)PowerfulcomputationalprogramscandeciphertheimagestogeneratesequencescomplementarytothetemplateDNAattheendofone“run,”andthesesequencesarethenmappedbacktothereferencegenomicsequence,toidentifyalterations.(Reproduced with permission from Metzker M: Sequencing technologies—the next generation, NatRevGenet11:31–46, 2010, © Nature Publishing Group.) looking for shared haplotypes, using single or a small number of SNPs that “tag” or identify a specific haplotype. Second, it is now possible to genotype millions of SNPs simultaneously, in a cost-effective way, using high-density SNP arrays, as described earlier.
Linkage analysis deals with assessing shared marker loci (i.e., SNPs) in family members exhibiting the disease or trait of interest, with the assumption that SNPs in linkage disequilibrium with the disease allele are transmitted through pedigrees. With time it becomes possible to define a “disease haplotype” based on a panel of SNPs, all of which cosegregate with the putative disease allele. Eventually, linkage analysis facilitates localization and cloning of the disease allele. Linkage analysis is most useful in mendelian disorders that are related to one gene with profound effects and high penetrance.