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*
• f*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*
• The*locaIon*of*the*SNP*locus*

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*

251mer* (25*nucleoIdes)*

!4# !3# !1# 0# 1# 3# 4# PMA* PMA* PMA* PMA* PMA* PMA* PMA* MMA* MMA* MMA* MMA* MMA* MMA* MMA* PMB* PMB* PMB* PMB* PMB* PMB* PMB* MMB* MMB* MMB* MMB* MMB* MMB* MMB* PMA* PMA* PMA* PMA* PMA* PMA* PMA* MMA* MMA* MMA* MMA* MMA* MMA* MMA* PMB* PMB* PMB* PMB* PMB* PMB* PMB* MMB* MMB* MMB* MMB* MMB* MMB* MMB* 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*