Ion AmpliSeq Technology
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1 Ion AmpliSeq Technology Dr. Franziska Freund Sr. Sequencing Sales Specialist The world leader in serving science
2 Ion AmpliSeq Technology: As Simple As PCR Your targets, your genome, your panel Comprehensive gene coverage with the lowest amount of DNA or RNA Input Simple As little as10 ng of DNA per pool FFPE-compatible PCR-based target selection Scalable Up to 24,000 primers per pool s of genes 96 barcodes for multiplexing Fast 1 day from DNA to results 2 hours to design custom panels 3.5 hours for target selection and library preparation 2
3 Broad Applications with Ion AmpliSeq Technology Ion AmpliSeq Technology Amplify Digest Ligate Exomes Gene Panels RNA Expression Fusions Community Panels CNV 3
4 4 Ion AmpliSeq Target Selection Overview
5 Technology Summary 5
6 AmpliSeq Designer Objectives Initial step of AmpliSeq workflow Repository for Panels and Designs Design creation for human and non-human organisms Development of highly specific primers Designs target genomic gene regions or SNP s Pre-design panels are: Ready-to-Use Community Easy to set up new designs Easy to share with collaborators FREE!!! 6
7 Key technology features: RNA pre-calculated designs DNA gene designs Standard AmpliSeq designs DNA hotspot designs 1-pool designs for SNP s Designs for Any Genome AgBio applications and more Design customization Sub-setting Mix and match Whitelist Support for Cell Free DNA (cfdna) designs 140bp amplicon sizes now available 7
8 Ion AmpliSeq Panels For DNA analysis and RNA expression measurement Ready-to-use Human Identity Panel Ready-to-use Human Ancestry Panel Ready-to-use RNA Apoptosis Panel Ready-to-use RNA Cancer Panel Ready-to-use Cancer Hotspot Panel Ready-to-use Comprehensive Cancer Panel Ready-to-use Inherited Disease Panel Community RNA Fusion Lung Cancer Research Panel Community BRCA 1 & 2 Research Panel Community Hearing Loss Research Panel Community CFTR Research Panel Community TP53 Research Panel Community AML Research Panel Community Cardio Research Panel* Community Dementia Research Panel Community Colon & Lung Cancer Research Panel 8 * The content provided herein may relate to products that have not been officially released and is subject to change without notice.
9 9 Ion AmpliSeq DNA panels
10 Ion AmpliSeq Cancer Hotspot Panel v2 As little as 10ng input 50 genes 207 amplicons Gene list available 10 For Research Use Only. Not for use in diagnostic procedures
11 Ion AmpliSeq Comprehensive Cancer Panel (CCP) Targets coding exons in 409 human oncogenes and tumor suppressor genes ~16,000 amplicons Detection of known COSMIC somatic mutations Gene list available 11 For Research Use Only. Not for use in diagnostic procedures
12 Ion AmpliSeq Exome 293,903 primer pairs across 12 primer pools >24,500-plex PCR! ~2.4 M PCRs per plate of 8 exomes!! Total DNA input as low as 50ng Covers >97% of CCDS (Release 12) >19,000 coding genes >198,000 coding exons (no UTRs, mirnas, or ncrnas) ~85% of human disease-causing variants found in coding regions or splice junctions Amplicon size range bp Average insert size is ~202 bp 12 For Research Use Only. Not for use in diagnostic procedures
13 OncoNetwork Global Consortia Prof. Harriet Feilotter Department of Pathology at Queen's University. Ontario Canada Prof. Ian Cree Warwick Medical School United Kingdom Marjolijn J.L. Ligtenberg, Arjen R. Mensenkamp Radboud University Nijmegen Medical Centre, The Netherlands Cecily P. Vaughn ARUP Institute for Clinical and Experimental Pathology Dr. Cristoph Noppen & Dr. Henriette Kurth VIOLLIER AG Basle, Switzerland Prof. Orla Sheils Trinity College Dublin, Ireland Prof Prof. Kazuto Nishio Faculty of Medicine, Kinki University Osaka, Japan Prof. Pierre Laurent Puig Université Paris Descartes, France Prof. Aldo Scarpa ARC-NET University of Verona Italy Dr. Ludovic Lacroix Institut Gustave Roussy Paris, France 13 Dr. Jose Costa IPATIMUP Medical Faculty of Porto. Portugal Dr. Nicola Normanno Centro Ricerche Oncologiche Mercogliano, Italy
14 Parallel DNA & RNA Analysis from single specimen 14 For internal use only, not for distribution
15 Oncomine Solid Tumour DNA Kit Application Sample type Input DNA required Genes Mutations Kit content Kit size Multiplexing SKU DNA somatic mutation detection (substitutions, insertions, deletions and inversions) extracted human DNA samples (including those from FFPE tissue) 10 ng or more. EGFR, ALK, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, MET, DDR2, KRAS, PIK3CA, BRAF, AKT1, PTEN, NRAS, MAP2K1, STK11, NOTCH1, CTNNB1, SMAD4, FBXW7, TP53 >1,800 cancer related mutations as supported by COSMIC database* Amplification oligonucleotides, library preparation reagents, x16 barcodes, DNA Equalizer 96 tests (split into 6 packs of 16 tests per pack) Possible to run up to 16 samples in a single run, as supported by sample bar coding A26761 * 15 For internal use only, not for distribution
16 Oncomine Solid Tumour Fusion Transcript Kit Application Sample type Input RNA required Genes Fusion Transcripts In-run + ve control genes Kit content Kit size Multiplexing SKU RNA fusion transcript detection extracted human RNA samples (including those from FFPE tissue) 10 ng or more of total RNA ALK, RET, ROS1, NTRK1 >75 targets of specific designs for cancer relevant fusions* + imbalance assay for non-targeted ALK fusions Amplification of housekeeping genes to show successful reaction and avoid false ves (MYC, ITGB7, LMNA, HMBS, TBP) Amplification oligonucleotides, library preparation reagents, x16 barcodes 96 tests (6 packs of 16 tests per pack) Possible to run up to 16 samples in a single run, as supported by sample bar coding A26762 *according to PubMed 16 For internal use only, not for distribution
17 17 Ion AmpliSeq RNA panels
18 From Single Gene Quantification to Discovery qpcr/ Digital PCR Expression levels for single gene targets Targeted Ion AmpliSeq Panels Panels for gene-level expression and fusion detection Ion AmpliSeq Transcriptome Differential gene expression/ profiling + sensitivity for lowabundance RefSeq genes Number and breadth of expression targets surveyed 18
19 Ion AmpliSeq RNA Cancer Panel 50 genes, from 500 pg unfixed RNA, one tube Targeted quantitative expression Complement to Cancer Hotspot Panel 19 For Research Use Only. Not for use in diagnostic procedures
20 Ion AmpliSeq Transcriptome Rapid, cost-effective gene-level expression analysis with simple workflows compatible with FFPE samples Survey precious or degraded samples with as little as 10 ng input RNA Perform gene-level expression surveys in less than 1 hour hands-on time (library prep), enabling RNA to gene expression levels in as little as 2 days Achieve better performance than arrays with wider dynamic range and detection of more genes at lower expression levels Obtain gene-level differential expression analysis with simple automated workflows that leverage existing microarray or NGS analysis pipelines 20 For Research Use Only. Not for use in diagnostic procedures
21 Ion AmpliSeq Transcriptome Overview Comprehensive coverage of RefSeq - 20,802 genes targeted Single primer pool simple workflow Reports at gene-level FFPE-compatible as little as 10 ng RNA Total RNA input from FFPE and other sources no selection or enrichment Assay design One amplicon per gene ~150 bp amplicon size with ~110 bp insert size Crosses exon boundary where possible Gene Isoform 1 Isoform 2 Isoform 3 Exon 1 Exon 2 Exon 3 Exon 4 Exon 1 Exon 2 Exon 4 Exon 1 Exon 2 Exon 3 21
22 22 Ion AmpliSeq Custom Design
23 Ion AmpliSeq Designer 23 *2.5 hours based on 10 kb design. A 250 kb design can take up to 48 hours.
24 Ion AmpliSeq Designer Your Panel > Choose Application Pick DNA or RNA Select a reference Give it a name 24
25 Ion AmpliSeq Designer Your Panel > Choose Application Adding targets Gene symbol Genomic coordinates dbsnp, COSMIC IDs Upload BED/CSV file 25
26 Choosing the Correct Design If the in silico coverage is sufficient this design is the best option For more coverage of GC-rich regions and tolerance for higher off-target 26
27 Ion AmpliSeq Designer Review your Design Before Ordering Your Panel > Choose Application Import it into a genome browser Coverage Summary Primer Details Amplicon ID, Fwd & Rev primer sequences Visualisein silico design in context with annotation tracks 27
28 Broad Applications with Ion AmpliSeq Technology Ion AmpliSeq Technology Amplify Digest Ligate Exomes Gene Panels RNA Expression Fusions Community Panels CNV 28
29 Start sequencing now at lifetechnologies.com/ampliseq For Research Use Only. Not for use in diagnostic procedures Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. TruSeq is a registered trademark of Illumina Inc. HaloPlex is a registered trademark of Agilent Technologies, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. 29
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