The Flu Network Astrid.Ferlinz@lifetech.com SMI Smittskyddsinstitutet ISS Istituto Superiore di Sanità CDC Centers of Disease Control JCVI J Craig Venter Institute USDA – NVSL US Dep of Agriculture – National Veterinary Service Labs SMI Stockholm Steve Glavas Picture JCVI Rockville David Wentworth Picture ISS Rome Gabriele Vaccari Picture CDC Atlanta Catherine Smith Picture Goals & Objectives: Evaluate & define protocol for Flu Typing Share & discuss results Make protocol/publication publicly available USDA – NVSL Ames, IA Mary Lea Killian Picture

Rapid Influenza A virus typing on the Ion PGMTM Sequencer RNA virus Genome size: approx 13.5 kb Comprised of 8 segments that encode up to 11 proteins Transcriptase: cap binding Transcriptase: elongation Transcriptase: protease activity? Haemagglutinin Nucleoprotein: RNA binding – transport of vRNA Neuraminidase: release of virus Matrix protein 2: Integral membrane protein – Ion Channel Non-structural protein 2: function not known Matrix protein 1: major component of virion Non-structural protein 1: RNA transport, translation, splicing For more information go to: http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html

PathAmpTM FluA Pre-Amplification Reagents PCR primer RT/PCR primer Core consensus 5’ Influenza A virus genomic RNAs Core consensus 3’ Highly specific Influenza A primer set (RT primer and PCR primer) High-fidelity master mixes for Reverse Transcription and PCR amplification of all 8 segments in a single tube Whole genome amplification Generates fragments that range in size from 900 bp to 2.4 kbp

Ion Torrent™ vs current workflow Current CE (Sanger) Throughput 10 samples/run (Ion 314™ chip) 1 sample/run Data Whole genome H and N genes only TAT 18 hrs/10 samples 2-3 Days Use RUO, research, epidemiology, monitoring Screening (subset of samples only) Specificity Sub-types, mixed infections Main H/N variants only; single infections Cost 115 Euro/sample* >€200 (H/N genes only) *excludes sample prep based on 10 multiplex on 314 chip Enables whole genome sequencing (WGS) of several specimen in a single run WGS permits deeper understanding of genetic landscape Identification of changes across the entire genome Much lower cost compared to CE-Sequencing Much shorter TAT for WGS compared to CE-Sequencing Deeper sequence coverage for detection of sub-types and mixed infections Improved sequence data for vaccines with higher specificity Ion PGM™ Sequencing allows: Deeper understanding of genetic landscape and re-assortments within the Influenza A genome

Workflow 60 min 3 hrs 30 min ~6 hrs 5 hrs 3 hrs RNA Extraction Reverse Transcription PCR Optional: Agarose gel analysis MagMax™-96 Viral RNA Isolation Kit 20 µl with up to 8 µl Sample 50 µl with ALL RT product 8 segments from 2400 to 900 bp size 30 min ~6 hrs 5 hrs 3 hrs Amplicon clean up & Quantitation DNA library prep Enrichment of library Sequencing MagMax™ Viral RNA Isolation Kit Nanodrop Ion Xpress™ Plus Fragment Library Kit Ion PGM™ 200 OneTouch™ Template Kit Ion 314™/316™ Chip

Results & Comparison with CE Sequencing Average Coverage Decrease of segment lengths Ion 316™ Chip Data provided by S Glavas, SMI, Stockholm Results from a 10-plex run using an Ion 316™ Chip Average coverage ranges from 636 (NS) to 5260 (M). This high coverage will allow much higher multiplexing. Customer feedback: 48-plex or even 96 should be possible There is higher coverage for shorter segments One explanation could be that the shorter segments are preferable amplified during the PCR. Clinical isolates (H1N1, 2009 H1 pandemic, H3) run in 10-plex on Ion 316™ Chip simultaneously Results were in 100% concordance with previous CE- data (H and N only) and were also confirmed by CE-sequencing of all 8 segments

Collaborator & internal R&D data Application note Released April 2013 Collaborator & internal R&D data

by collaborators from SMI Data analysis plugin Launched in IC in April 2013 by collaborators from SMI

Flu Season Winter/Spring 2013 PathAmpTM FluA Pre-Amplification Reagents were used to sequence whole-genome of H7N9 Highly accurate and sensitive results from both swab samples and isolated virus samples Detection of mixed infections Publication submitted Sequencing of the H7N9 virus during China Outbreak Flu Season Winter/Spring 2013

Start sequencing now at lifetechnologies.com/iontorrent Thank you ! Start sequencing now at lifetechnologies.com/iontorrent

Limitations and Disclaimers For Research Use Only. Not for use in diagnostic procedures. Limitations and Disclaimer: Life Technologies Corporation takes no corporate position on the use of selection methods in IVF and prenatal settings though we acknowledge that people disagree about its appropriate use and it should ALWAYS be provided with full and informed, non-coerced prior informed consent. The PGM™ System and equipment used herein is RUO marked and may not be GMP. The results shown may not represent actual performance in an IVF or any other setting. LTC does not assure or endorse the use of its methods in ANY clinical setting outside of those that have been reviewed by the FDA or similar oversight body. © 2013 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