1. The world leader in serving science Sanger sequencing using Ion AmpliSeq™ primers and libraries Stephen Jackson, Ph.D. Associate Director, Product Applications GSD

2. 2 Extracting maximal information from minimal sample amounts Problem: Low amounts of material extracted from samples Need orthogonal method for confirming minor alleles Can Sanger sequencing on CE be used with Ion AmpliSeq amplicon designs and prepared libraries to enhance the analysis of minor alleles in small amounts of sample? Opportunity: Ion AmpliSeq™ panels have been optimized to target oncogenic alleles from low input amounts of starting material

3. 3 Prepare samples using standard PCR techniques – simple and fast Starting with just 10 ng of input DNA – compatible with FFPE samples May be able to use less Ready-to-Use panels, build your own Custom panels, or leverage Community panels >24,000-plex PCR amplification! Ion Ampliseq™ workflow Ion AmpliSeq Pool – source of amplified target sequences Ion AmpliSeq Library – source of amplified target sequences

4. 4 M13 sequences must be added to Ion AmpliSeq primers M13 forward: 5’-TGTAAAACGACGGCCAGT-3’ M13 reverse: 5’-CAGGAAACAGCTATGACC-3’

5. 5 Sanger sequencing confirmation from an Ion AmpliSeq pool

6. 6 Confirming Ion AmpliSeq frequencies by Sanger sequencing TP53_10.1.470 G>TTP53_11.1324631 G>CTP53_8.384088 T>C FFPE 521.8%20.2%17.9% Results obtained by NGS Results confirmed by Sanger Sequencing of the Ampliseq pool Traces are from Sequence Scanner software

7. 7 Minor Variant Finder: a key innovation for detecting rare variants 1:101:1 variant : normal 1:20 1:4 Sanger sequencing can detect variant alleles in a DNA sample 50% Frequency: Variant detectable and called as ambiguous base 20% Frequency: Variant detectable but not called 5%-10% Frequency: Variant not detectable above background

8. 8 Minor Variant Finder: a key innovation for detecting rare variants Minor Variant Finder software: 1. Determines background peaks in control sample run concurrently with test sample 2. Compares and removes background peaks from the test sample 3. Looks for variants at identical position in forward and reverse sequencing reactions 4. Calculates area under the peak to determine allele frequency

9. 9 MVF correlation with NGS allele frequencies is high Correlation with NGS allele frequencies is about the same as the correlation between forward and reverse reactions Allele frequency CE (%) Allele frequency NGS (%) Allele frequency CE forward (%) Allele frequency CE reverse (%)

10. 10 Three scenarios for variant detection and confirmation Confirm variants using amplicons in Primer Designer database Confirm variants by Sanger sequencing using Ion AmpliSeq™ primers Confirm variants by Sanger sequencing the Ion AmpliSeq™ pool or library

11. 11 Conclusions Sanger sequencing results can be obtained using Ion AmpliSeq™ library primer sequences or from existing Ion AmpliSeq™ library pools Robust genotyping results using both Ion AmpliSeq™ next- generation sequencing (NGS) and confirmatory Sanger sequencing can be generated from less than one nanogram of FFPE DNA Previously sequenced NGS libraries can be used as a direct input for confirmatory Sanger sequencing

12. 12 Arpad Gerstner Edgar Schrieber Kamini Varma Ion Torrent Applications Group Arpad Gerstner Edgar Schrieber Kamini Varma Ion Torrent Applications Group Acknowledgements For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. If you are using Capillary Electrophoresis instruments and are interested in contributing to the newest advances, my colleagues would like your to speak with you. Please see Stephane Jankowski or myself at the end of this satellite meeting.

13. The world leader in serving science Thank you