www.agilent.com/genomics/surefish Latest Innovation and Advancement in High Resolution Oligo FISH Technology Nargol Faravashi – Sr. Product Manager, Genomics, Agilent Technologies Shashikant Kulkarni M.S (Medicine), Ph.D., FACMG Head of Clinical Genomics Medical Director Cytogenomics and Molecular Pathology Associate Professor of Pediatrics, Genetics, Pathology and Immunology Washington University School of Medicine, St Louis Overview FISH Technology: • Method for detection of translocations, inversions, amplifications, deletions and duplications • Better detection of low-level mosaicism • Enables Visualization in intact cellular context • positional information of aberrations in chromosomal context Limitations of current technologies: Clone based methodologies: bacterial artificial chromosomes (BAC), fosmids, PAC (P1-derived artificial chromosome), YAC (yeast artificial chromosome), PCR templates • Resolution limitations (150-300 Kb) • Not ideal for visualization/verification of smaller genomic regions • Labor intensive • Rapid turnaround time Agilent Industrial Scale Manufacturing: Oligo Library Synthesis (OLS) Oligo Library Synthesis Solution of user-defined oligonucleotides: Release “Oligo Library” oligonucleotides from the microarray BioReagent: substrate .Gene(ome) synthesis .Gene silencing library .Genome partitioning .OligoFISH Oligos specifically selected to unique sequences Repetitive elements Segmental duplications Step 1: Tile region with long oligonucelotides: Step 2: Remove any non-unique oligos: Step 3: Manufacture labeled probes using specifically designed long oligonucleotides Unique design methodology for specific targets , provides unparalleled resolution with high sensitivity and specificity Agilent’s SureFISH offerings SureFISH Probes: 5µl configuration • ASR manufactured under cGMP/QSR guidelines General Purpose Reagents: 100µl configuration • Hybridization buffer • Mounting buffer • Wash buffer1 • Wash buffer2 www.agilent.com/genomics/surefish Agilent’s SureFISH offerings Integration with Microarray Data: • Characterization of CGH findings by visualizing the probe of interest in Agilent CytoGenomics Software • Link from CytoGenomics software to eCommerce website http://ft2.genomics.agilent.com/ProductSearch. aspx?chr=1&PageID=3000 Superior Performance & Key attributes : Coverage of Most Relevant Targets Broad menu-Hundreds of relevant probes for constitutional and cancer applications Flexibility • Various fluorescent dyes to fit visualization needs • In silico design methodology to target specific regions Quality Control • Probe localization to correct chromosomes demonstrated by FISH hybridization images. Images provided on the website for both 4, 14 hour, metaphase & interphase • Each probes is sequence verified to ensure specificity to the region of interest Streamlined Workflow • Pre-labeled probes reduce labor time • Compatible with established FISH procedures and filter sets • Fast Hybridization time – 4 hours • No Cot-1 DNA Comprehensive SureFISH Probe Menu Telomere Telomere Telomere dLOC100130417,SAM FAM110C,SH3YL1,ACP1 CHL1,, CNTN4,, D11,NOC2L,KLHL17, ,FAM150B, SYN2,TIMP4,PPARG PLEKHN1,C1orf170,H MYCNOS,MYCN, ,, ES4,ISG15,AGRN,RN NCOA1, LBH,YPEL5,, TSEN2,LOC1001294 F223,C1orf159,LOC25 ABHD12,GINS1,NANP,N 80,MKRN2,, 4099,MIR200B,MIR20 INL,PYGB,ZNF337, CEP19,FBXO45,LRR Telomere 0A,MIR429,TTLL10, EML4,COX7A2L,, C33,PIGX,WDR53,, RPL23AP53,ZNF596, SKI,MORN1,LOC1001 VRK2,FANCL,, BCL11A,, FOXL2,C3orf72,, GATA4,NEIL2,, Telomere 29534,RER1,PEX10,P FLJ16341,REL,PUS10 NULL,, MECOM,, Telomere PNOC,ZNF395,, C9orf66,DOCK8,KAN LCH2,PANK4,HES5,L KIAA1841,LOC339803,C BDH1,, DDHD2,PPAPDC1B, K1,, DMRT1,DMRT3,, Telomere FAM20C,, OC115110,LOC10013 2orf74,AHSA2,USP4 PIK3CA,KCNMB3,, WHSC1L1,LETM2,FG RCL1,MIR101- PLEKHG4B,LRRC14B,C Telomere LOC100133311,HOX 3445,TNFRSF14, MALL,NPHP1,LINC0011 SOX2-OT,SOX2,, FR1,C8orf86,, 2,JAK2, CDC127,SDHA, TERT, FOXC1,GMDS, A5,HOXA6,HOXA7,H AJAP1, 6, MBD5,, SST,RTP2,LOC1001 CEBPD,LOC1002878 C9orf46,CD274,, KHDRBS2, C6orf15,PSORS1C1,C OXA10- AMPD1,NRAS,CSDE1 HOXD13,HOXD12,HOXD 31635,BCL6,, Telomere 46,KIAA0146,, CHD7, PAX5,MIR4540,MIR44 DCBLD1,ROS1,VGLL2,R DSN,PSORS1C2,CCH HOXA9,HOXA9,MIR1 ,SIKE1, NOTCH2, 11,HOXD10,HOXD9,HO CWH43,FRYL,OCIA ZNF721,PIGG,PDE6B, RUNX1T1, TRPS1,, 76,, OS1, APC, CR1,TCF19,POU5F1,P 96B,HOXA10,HOXA1 Centromere XD8,LOC100506783,MIR D1,OCIAD2,SLC10A ATP5I,MYL5,MFSD7,P EXT1, NULL,, PTCH1,LOC10050734 KDM3B,REEP2,EGR1,E SORS1C3, 1,HOXA11- RNF115,CD160,PDZK 10B,HOXD4,HOXD3,LO 4,ZAR1,, CGF3,LOC100129917, MYC,PVT1,MIR1204,, 6, LMX1B,, TF1, SUPT3H,RUNX2,, AS1,HOXA13, 1,, BCL9,, C401022,HOXD1,LOC40 PIGX,PAK2,SENP5, CPLX1,, PVT1,MIR1204,, ASS1,LOC100272217 PDE6A,SLC26A2,TIGD6, MTRNR2L9,KHDRBS2, IKZF1,FIGNL1,DDC, GJA5,GJA8,, 1022,, NCBP2,LOC152217, FAM53A,SLBP,TMEM ZNF251,ZNF34,RPL8, ,FUBP3,PRDM12,EX HMGXB3,CSF1RTCOF1, , EGFR, VOPP1, HIP1, ANXA9,CERS2,FAM6 OSGEPL1,ORMDL1,PM PIGZ,MFI2,MFI2- 129,TACC3,FGFR3,LE ZNF517,ZNF7,COMM OSC2,ABL1,QRFP,FI CD74, CEBPD,KIAA0146,LOC LOC100506136,SHF 3A,PRUNE,SETDB1, S1,, PAX3,, CCDC140,, AS1,DLG1,MIR4797, TM1,WHSC1, D5,ZNF250,ZNF16,ZN BCD1,, RANBP17,TLX3,MIR391 100287846,, MYB, M1, TES, MET, BRAF, ISG20L2,RRNAD1,M HDAC4,MIR4440,MIR44 LOC100507086,, RPL21P44,CHIC2,GS F252,TMED10P1,C8o NOTCH1,MIR4673,MI 2,NPM1,FGF18MIR3912, MLLT4,, MRPS33,LOC100507 RPL24,HDGF,PRCC, 41,, LOC100505806,LOC X2,, PDGFRA, KIT,, rf77,C8orf33 R4674,, NPM1,FGF18, FAM120B,MIR4644,PS 421, EPHB6,TRPV6, SH2D2A,NTRK1,INSR HDAC4,MIR4440,MIR44 285692,SEMA5A,SN TET2,TET2,,, Telomere EHMT1,FLJ40292,MI CNOT6,SCGB3A1,FLT4, MB1,TBP,PDCD2 GALNT11,MLL3, R,PEAR1,LRRC71,AR 41,MGC16025,MIR4269, ORD123,TAS2R1,, Telomere R602 BTNL8,BTNL3,BTNL9,O Telomere RBM33,SHH, VIPR2 HGEF11,MIR765,, MIR2467,, KIAA0226,KIAA0226, Telomere Telomere RALGPS2,FAM20B,T ING5,D2HGDH,GAL3ST MIR922,FYTTD1,LR R2V2,TRIM7,MIR4638,T OR3A,ABL2,, DISP1,, 2,NEU4,PDCD1,C2orf85, CH3,IQCG,RPL35A,L RIM41,GNB2L1,SNORD9 Telomere FH,KMO,, Telomere MLN, 6A,SNORD95,TRIM52. YWHAE,CRK,MYO1C SDCCAG8,AKT3,AKT Telomere Telomere PAFAH1B1,KIAA0664, 3,AKT3, SHBG,SAT2,ATP1B2,T SH3BP5L,MIR3124,Z P53,WRAP53,EFNB3,D NF672,ZNF692,PGB2 NAH2, ALOX15B,ALOX12B,MI Telomere Telomere R4314,ALOXE3,HES7, INS- Telomere PER1,VAMP2,TMEM10 IGF2,IGF2,MIR483,IGF2- IQSEC3,LOC574538,S 7,MIR4521,MIR3676,C1 Telomere AS,INS,TH,MIR4686, LC6A12,SLC6A13, 7orf59,AURKB,LINC003 Telomere ZMYND11,DIP2C,FLJ KCNQ1, WT1,WT1-AS, A2M,C12orf33,LOC144 Centromere 24,CTC1,PFAS, Telomere EXT2,, NULL, TRAP1,CREBBP, RBFOX1, 45983,GATA3, 571,PZP, Centromere SNRPN,SNURF,SNURF, PMP22,PMP22,PMP22, USP14,THOC1,COLE CCND1,ORAOV1,FGF19 TNFRSF17,SNX29, C10orf114,MIR1915,C CASC1,LYRM5,KRAS, FOXG1,C14orf23, SNORD107,PAR- MIR4731,, TEKT3, C12, SS18,, TCF4, , ORAOV1,FGF19, SN,PAR5,SNORD64,SN C16orf45,KIAA0430,NDE1, 10orf140,MLLT10,DN R3HDM2,INHBC,INHB Centromere METTL21D,SOS2,L2HG CDRT4,FAM18B2- BCL2,KDSR,, FGF4,FGF3, ORD108, UBE3A, MIR484,MYH11, AJC1,, NULL,, RET,, E,GLI1,ARHGAP9,MAR MPHOSPH8,PSPC1T, DH,ATP5S,CDKL1, NIN, CDRT4,FAM18B2, BCL2,KDSR,VPS4B, YAP1,BIRC3,BIRC2,TME OCA2,, OTUD7A, FOPNL,ABCC1,ABCC6,, BMPR1A,, S, TTL,, ELF1,WBP4, PTGDR,PTGER2, RAI1,SMCR5,SREBF1, HSBP1L1,TXNL4A,RB M123, NPAT,ATM, CHRNA7, C16orf52,VWA3A, ACTA2,FAS,FAS- MBD6,DCTN2,KIF5A,PI DLEU2,TRIM13,KCNRG, SAMD4A, MIR33B, SPECC1, FA,ADNP2,LOC10013 ZBTB16, ANKRD26P1,C16orf87,D ATXN2L,TUFM,MIR4721,S AS1, DPCD,FBXW4, P4K2C,DTX3,ARHGEF MIR16- C14orf33,KTN1, MIR4733,NF1,OMG,EVI 0522,PARD6G CD3E,CD3D,CD3G,UBE NAJA2,GPT2,MYLK3,NE H2B1,ATP2A1,RABEP2,CD CALY,PRAP1,C10orf1 25,SLC26A10,B4GALN 1,MIR15A,DLEU1, RAD51B, 2B,EVI2A,RAB11FIP4, Telomere 4A,ATP5L,MLL, TO2,ORC6,SHCBP1,VP 19,NFATC2IP,MIR4517,SP 25,ECHS1,MIR3944,P T1, 9- DLEU7, ZIC5,ZIC2, DICER1,MIR3173,DICE ACACA, CD3D,CD3G,UBE4A,AT S35, SMAD6, NS1,LAT, AOX,MTG1,SPRN,LO Mar,AGAP2,CDK4,CYP PCID2,CUL4A,LAMP1,G R1-AS, TCL6,TCL1B PGAP3,ERBB2,MIR472 P5L,MLL,TTC36,TMEM2 CYP1A1,CYP1A2,CSK,M ZNF668,ZNF646,PRSS53,V C619207,CYP2E1,SY 27B1,LOC100130776, RTP1, RASA3,CDC16 NULL, 8,MIEN1,GRB7,, 5,IFT46,ARCN1,PHLDB1 IR4513,LMAN1L,CPLX3, KORC1,BCKDK,KAT8,PRS CE1,SPRNP1 METTL1,METTL21B,T Telomere Telomere DHX8,ETV4, Telomere , SFM,TSPAN31, ULK3,SCAMP2, S8,PRSS36, MAF, CRHR1,LOC100128977 Centromere MLL,TTC36,TMEM25,IFT RPSAP52,HMGA2, LOC100507217,CHD2,MI LOC400550,FOXF1,MTHFS ,IMP5,MAPT,LOC10013 CLTCL1, HIRA,, 46,ARCN1,PHLDB1,TRE RAP1B,SNORA70G,LO R3175, D,FLJ30679,FOXC2,FOXL1 0148,STH,KIAA1267, TBX1,GNB1L,, H, SNX19, C100507250,NUP107, IGF1R,MIR4714,PGPEP Telomere MKS1,LPO,MPO,BZRA TXNRD2,COMT,MI JAM3,NCAPD3,VPS26B, SLC35E3,MDM2,CPM, 1L P1,LOC100506779,MIR R4761,ARVCF THYN1,ACAD8,, NULL RPL6,PTPN11,, Telomere Telomere 142,MIR4736, SOX9, CRKL,AIFM3,LZTR Telomere TBX5,LOC255480, STS, CDKL5,RS1, ASPSCR1,STRA13,LR 1,THAP7,FLJ39582, WDR66,BCL7A,MLXIP DMD, RP2,PHF16,, RC45 MIR650,IGLL5, Telomere SLC38A5,FTSJ1,P Telomere IGLL5,RTDR1,GNA ORCN,EBP,TBC1D Telomere Z,RAB36,BCR,FBX 25, PPAP2C,MIER2,THE Telomere W4P1,CES5AP1,ZD GLOD5,GATA1,HD BCORP1,T G,C2CD4C,SHC2,OD JAG1, HHC8P1,IGLL1,C22 Centromere AC6,ERAS,PCSK1 XLNG2P,T F3L2,MADCAM1,C19 DNMT3B,MA orf43,GUSBP11,RG CLIC6,LINC001 N, XLNG2P,T orf20,CDC34,GZMM, PRE1, L4,ZNF70,VPREB3, 60,LOC1005063 BCYRN1,GJB1,ZM XLNG2P,C POLRMT,FGF22,RNF PTPRT C22orf15,CHCHD10 85,RUNX1, YM3,NONO,ITGB1 Yorf15B,K 126,FSTL3,PRSS57, ABCC13,HSP ,MMP11,SMARCB1, LINC00114,LIN BP2, TSIX,XIST, STK11,C19orf26,ATP A13,LIPI,RBM DERL3,SLC2A11,L DM5DTelo C00114,LINC00 PLP1,RAB9B,, 5D,MIDN,C19orf23,CI 11,SAMSN1, OC284889,MIF,GST mere 114,ETS2,PSM COL4A6, RBP,C19orf24, C20orf108,AU T2,GSTT2B,DDTL, G1,BRWD1, KCNE1L,ACSL4, LYPD4,DMRTC2,RPS RKA,CSTF1, DDT,GSTT2,GSTTP LINC00323,MIR