Tumor Hotspot MASTR Plus Research Application For the use of somatic and germline variant detection of selected target regions in 26 frequently mutated genes in: – FFPE-derived DNA – FFT- and blood-derived DNA A molecular research panel for the identification of SNVs in the hotspots of 26 frequently mutated genes in solid tumors. This NGS assay is designed with input from selected INCa centers in France. Assay Characteristics Genes with hotspots included AKT ERBB2 (HER2) IDH1 PDGFRA ALK ERBB4 IDH2 PIK3R1 BRAF FGFR2 KIT PIK3CA CDKN2A (p16-INK4A, p14-ARF) FGFR3 KRAS PTEN (full gene coverage) CTNNB1 (β-catenin) H3F3A (Histone H3, F3A) MEK1 (MAP2K1) STK11 (LKB1) (full gene coverage) DDR2 HIST1H3B (Histone H1, 3B) MET EGFR HRAS NRAS Assay Characteristics Cont. Genomic region analyzed Number of amplicons Amplicon length Number of plexes Verified with NGS system Designed to be compatible with Complete variant spectrum Illumina MiSeq reagent kit v2 25.7 kb 252 128-245 bp 4 Illumina MiSeq Illumina NextSeq, MiniSeq and Ion Torrent NGS Systems SNVs Illumina MiSeq reagent kit v3 Sequencer capacity Total reads 12,000,000 22,000,000 # samples/run @ 5% VAF sample 20 reads per allele** 17 31 # samples/run @ 50% VAF sample 20 reads per allele** 170 308* *only 192 MID combinations available ** Number of samples per run for Illumina & IonTorrent NGS Systems can be calculated via the sequencing calculator. Performance 98.8 % On target read count >97 % DNA input as low as ng per plex 100% Percentage of amplicons Uniformity of amplification (0.2x mean coverage) 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5 x mean coverage Graph presenting the read counts of Tumor Hotspot MASTR Plus amplicons, showing their uniform representation. √ ALK Exon 20 to 29, includes mutations in kinase domain affecting Ile1171, Phe1174, Leu1196, Phe1245, Gly1269, Arg1275 and Tyr1278 √ Exon 11 and 15, includes mutations in kinase domain affecting Gly466, Gly469, Asp494, Val600 and Lys601 √ CDKN2A (p16-INK4A, p14-ARF) Full exon coverage √ CTNNB1 (β-catenin) Exon 3 includes mutations affecting Asp32, Ser33, Gly34, Ser37, Thr41 and Ser45 √ DDR2 All coding exons from exon 4 to 19 √ EGFR Exon 18 to 21, spanning the kinase domain that includes mutations affecting Glu709, Gly719, Glu746-Pro753, Ser768 and Leu858 √ ERBB2 (HER2) Exon 19 to 21, spanning the kinase domain that includes mutations affecting Leu755, Gly776, Val777 and Val842 √ ERBB4 Exon 10 and 12 √ FGFR2 Exon 7, 12 and 14, including mutations affecting Ser252, Asn549 and Lys659 FGFR3 Exon 7, 9, 14 and 16, including mutations affecting Arg248, Ser249, Tyr373 and K650 √ H3F3A (Histone H3, F3A) Exon 2, including mutations affecting Lys28 √ HIST1H3B (Histone H1, 3B) Exon 1, including mutations affecting Lys28 √ HRAS Exon 2-4, including mutations affecting Gly12, Gly13 and Gln61 √ IDH1 Exon 4 including mutations affecting Arg132 √ IDH2 Exon 4 including mutations affecting Arg140 and Arg173 √ BRAF KIT KRAS √ √ √ √ √ MET Exon 2, 10, 14 to 20, including mutations Glu168, Thr1010 and the kinase domain including Tyr1253 √ NRAS Exon 2-4, including mutations affecting Gly12, Gly13 and Tyr61 √ PDGFRA Exon 12, 14 and 18, including mutations in the kinase domain affecting Asp852 PIK3R1 Exon 11-13, including mutations affecting aa 452 to 464 PIK3CA Exon 2, 3, 10, 11 and 21, including mutations affecting Glu39, Arg88, Glu542, Glu545 and His1047 PTEN Full exon coverage STK11 (LKB1) Full exon coverage √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ Exon 2 to 3, including mutations affecting Lys57 and mutations in Pro124 in the kinase domain √ √ Exon 8 to 11, 13, 14, 17 and 18, including mutations affecting aa417-419, 557-560, Leu576 and kinase domain mutations √ √ √ √ Exon 2 to 4, including mutations affecting Gly12 and Gly13 MEK1 (MAP2K1) √ Pancreas, thyroid, prostate, glioblastoma, and others √ Blood Breasts/ovarians √ Stomach Colon Exon 4, includes mutations in the PH domain affecting Glu17, Phe35 Coverage and included hotspot mutations Skin Lungs AKT Gene √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ MASTR Plus Workflow Multiplex PCR Plan run Multiplex PCR Mixing Clean up Quality Control Mixing plexes of single sample Universal PCR Clean up Dilution Purification Pooling NGS SEQUENCING Universal PCR Quality Control Ordering Information Cat. No. Product Name Sample MR-0200.024 Tumor Hotspot MASTR Plus 24 • MID (Molecular Identifiers) kits are necessary to complete the workflow www.agilent.com Not for EU [email protected] For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice. PR7000-0722 © Agilent Technologies, Inc. 2017 Published in the USA, October, 2017 5991-8368ENUC Purification Equimolar pooling DATA ANALYSIS