Genotyping*Technology* 021223*How*to*Analyze*Your*Own* Genome* - PDF document

Genotyping*Technology* 021223*How*to*Analyze*Your*Own* Genome* Fall*2014* How*do*we*know*what*SNPs*someone* has?* 1. Sequence*enIre*genome* – Expensive*lots*of*info*we*may*not*need* 2. Genotype*using*Arrays*

What*is*an*Array?* • Look*for*specific*known*SNPs* • Use*binding*features*of*DNA*to*do*this*quickly* and*efficiently* Southern*Blot* hVps://www.mun.ca/biology/scarr/Gr12118.html*

Arrays*blot*on*Silica* Why*Silica?* • Easy*to*fabricate* • Lots*of*tools*for*placing*small*things*in* different*places* • Low*background* • Can*measure*electrical/visual*signals*easily*

DNA*(probes)*on*surface* DNA*on*beads*embedded*in*wells*

What*arrays*have*on*them* • Specific*SNPs* • Custom*SNPs* • Both* • New*SNPs*as*Ime*goes*on* SNP*Genotyping*with*SNP*Array* • An*array*of*oligonucleoIde*sequences*is*laid* across*the*surface*of*the*chip.** • The*sample’s*DNA*is*amplified,*and*hybridized*to* the*array.** • The*array*is*scanned*to*quanIfy*the*relaIve* amount*of*sample*bound*to*each*feature.* • For*SNPs,*there*is*a*pair*of*probes:*one*for*each* of*the*alleles.*

SNP*Array*Technology:*Affymetrix*Array* • The*fragment*of*DNA*harboring* an*A/C*SNP*to*be*interrogated* by*the*probes* • 251mer*probes*for*both*alleles* 251mer* • The*locaIon*of*the*SNP*locus* (25*nucleoIdes)* varies*from*probe*to*probe* The*DNA*binds*to*both*probes* • regardless*of*the*allele*it*carries* Mismatch*binds*less*Ightly* • • Fewer*bound*at*end*of* experiment* • Fewer*DNA*means*dimmer* signal* !4# !3# !1# 0# 1# 3# 4# PM A * PM A * PM A * PM A * PM A * PM A * PM A * MM A * MM A * MM A * MM A * MM A * MM A * MM A * PM B * PM B * PM B * PM B * PM B * PM B * PM B * MM B * MM B * MM B * MM B * MM B * MM B * MM B * PM A * PM A * PM A * PM A * PM A * PM A * PM A * MM A * MM A * MM A * MM A * MM A * MM A * MM A * PM B * PM B * PM B * PM B * PM B * PM B * PM B * MM B * MM B * MM B * MM B * MM B * MM B * MM B * Purple*for*sense,*Blue*for*anIsense*strands*of*DNA**

SNP*Array*Technology:*Illumina*Array* • The*fragment*of*DNA*harboring*an* SNP*(A/C)*to*be*interrogated*by*the* probes* • AVached*to*each*Illumina*bead*is*a*501mer* sequence*complementary*to*the*sequence* adjacent*to*the*SNP*site.** • The*single1base*extension*(T*or*G)*that* is*complementary*to*the*allele*carried* by*the*DNA*(A*or*C,*respecIvely)*then* binds*and*results*in*the*appropriately1 colored*signal*(red*or*green,* respecIvely)* • Ligase*enzyme*links*the*new*base*to* the*strand*on*the*bead* • Can*only*disInguish*between*A/G*C/T*

Calling*Genotypes* • The*raw*signal*intensiIes* from*the*SNP*array*can*be* noisy* • How*to*cope*with*the* noise* – Pool*the*raw*signal* intensiIes*from*mulIple* individuals*for*each*SNP* and*perform*a*cluster* analysis* – Three*clusters*for*each*of* the*three*possible* genotypes*(AA,*AB,*BB)* Each*dot*represents*the*raw* signal*intensity*for*a*SNP*for* each*individual*

Also*care*about*copy*numbers* • How*might*we*look*at*these?*

Summary* • Can*uIlize*DNA*binding*to*idenIfy*changes*in* genes* • Genotyping*by*Array*is*very*fast*and*efficient*