Ion PGM Heterogeneity * 1 * 1 * 2 * 2 * 2 * 2 * 2 * 1 *1 * 2 DNA DNA DNA ArcturusXT LCM Laser Capture Microdissection LCM A Workflow for Cancer Profiling Applied Biosystems ViiA7 PCR qpcr QC Assay Ion PGM Ion AmpliSeq Cancer Hotspot Panel v2 Applied Biosystems ViiA7 PCR TaqMan Mutation Detection Assays ASP LST A ASB G ASP Allele-specific primer ASB Allele-specific blocker*(mgb) LST Locus-specific TaqMan probe LSP Locus-specific primer Quant Studio 3D PCR 1. 2. DNA QC 3. 4. LSP Heterogeneity Heterogeneity FFPE LCM DNA NGS DNA 1 Ion PGM LCM Applied Biosystems Arcturus XT LCM Applied Biosystems Arcturus XT IR UV Digital PCR dpcr by TaqMan SNP Genotyping Assays IR UV
Ion PGM Library builder Ion Onetouch 8 Multiplex sample 96 384 1 LCM LCM 500 microns 500 microns 2 LCM Ion AmpliSeq Cancer Hotspot Panel v2 10ng DNA 50 207 KRAS BRAF EGFR TP53 50 2800 154bp FFPE DNA FFPE DNA FFPE LCM FFPE DNA DNA DNA LCM DNA PCR NGS 1 HE FFPE LCM 1. LCM FFPE 2 2.4mm 2 2.2mm 2 70% [ ] PEN Membrane Glass Slides LCM0522 FFPE 10 HE [ ] Applied Biosystems Arcturus XT CapSure Macro LCM Caps LCM0211 Arcturus PicoPure DNA Extraction Kit KIT0103 DNA DNA DNA
2. DNA QC PCR qpcr Human Genomic DNA 1 RNase P Long Amplicon 256 bp Primer TaqMan Probe RNase P Amplicon 87 bp Assay TaqMan RNase P Detection Reagents 4316831 DNA Long Amplicon Short Amplicon 87 bp Long Amplicon 256 bp DNA PCR : ViiA 7 PCR : TaqMan Gene Expression Master Mix : standard mode Primer & Probe : Primer 900 nm Probe 250nM : 20uL/well DNA 1uL/well Replicates: n=3 Human Control DNA 10ng/ ul 4 5 DNA TaqMan Assay ION070- A1302IH A Control DNA 3 AHuman Control DNA Short Amplicon 87bp Long Amplicon 256bp FFPE Long Amplicon Short Amplicon DNA 3 B B Short Short Amplicon 87 bp Long Amplicon 256 bp Amplicon 1.309ng / Quantity Quantity ul0.553ng/ul Sample ng/ul SD ng/ul SD Human Control DNA Long Amplicon 256bp RQ 0.064 1.309 0.023 0.151 0.012 0.034 4 RQ DNA 0.553 0.018 0.033 0.003 NGS 3 PCR qpcr A Human Control DNA 87bp 256 bp FFPE Long B PCR DNA
3. NGS Ion AmpliSeq Cancer Hotspot Panel v2 Input 9 L Short Amplicon 11ng 5ng Template Input DNA Input DNA 10ng 5 100 Coverage 99 Reads Variant 1.2 1.0 1.000 Ion AmpliSeq Cancer Hotspot Panel v2 Ion AmpliSeq Library kit 2.0 Ion PGM Template OT2 200 kit Ion PGM Sequencing 200 kit v2 RQ 0.8 0.6 0.4 Chip: Ion 316 Chip 2 Torrent Suite3.2 Variant caller 3.2.4 0.2 0.0 Human Control DNA 0.064 0.034 4 Human Control DNA Long Amplicon 256 bp A 20X Coverage 100X Coverage Reads on target Depth Reads# 100% 99.17% 94.41% 1551 386K 100% 99.61% 88.69% 3939 1048K B 5 A BPhred Score
4. PCR qpcr NGS Variant PCR qpcr TaqMan Mutation Detection Assay 1copy Assay 10 7 copy wild type 1copy mutation 0.1% 46 778 TaqMan Mutation Detection Assay 6 QuantStudio 3D PCR 0.01% Allele1 specific PCR ASP*1 LST T A LSP ASB2 MGB G GASB2 A 46 6. TaqMan Mutation Detection Assay Allele2specific PCR ASP*2 LST C G LSP ASB1 MGB A AASB1 G ABL1 CSF1R FGFR3 KDR NRAS SMARCB1 AKT1 CTNNB1 FLT3 KIT PDGFRA SMO ALK EGFR GNAS KRAS PIK3CA SRC APC ERBB2 HNF1A MET PTEN STK11 ATM ERBB4 HRAS MLH1 PTPN11 TP53 BRAF FBXW7 IDH1 MPL RB1 VHL CDH1 FGFR1 JAK2 NOTCH1 RET CDKN2A FGFR2 JAK3 NPM1 SMAD4 FFPE LCM LCM FFPE DNA PCR Long amplicon Short amplicon Long amplicon Short amplicon RQ DNA RQ 0.2 NGS FFPE PCR NGS NGS Variant NGS PCR 7. Ion PGM Variant Heterogeneity 2013 2
Heterogeneity 3200 30 45 1970 43 56 1981 B HBV 30 C B 30 40 Pathway Driver/Promoter Target CT MRI Genome Analysis Center-GAC 3200 B 9000 C 30 Ordering information ArcturusXT IR & UV Applied Biosystems ViiA 7 PCR TaqMan Array Ion Personal Genome Machine QuantStudio 3D PCR ARCTURUSXT-B VIIA7-01 PGM-150M QS3D-02 Applied Biosystems Arcturus XT LCM 0120-477-392 Facebook & Twitter facebook.com/lifetechnologiesjapan@lifetechjpn The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. 2013, Life Technologies Japan Ltd. All rights reserved. Printed in Japan. ION071-B1304IH www.lifetechnologies.com/tc 108-0023 4-2-8 564-0052 10-28 TEL.03 6832 9300 FAX. 03 6832 9580 TEL.06 6389 1201 FAX. 06 6389 1206 www.lifetechnologies.com