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The Flu Network SMI Stockholm Steve Glavas Picture ISS Rome Gabriele Vaccari Picture CDC Atlanta Catherine Smith Picture JCVI.

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Presentation on theme: "The Flu Network SMI Stockholm Steve Glavas Picture ISS Rome Gabriele Vaccari Picture CDC Atlanta Catherine Smith Picture JCVI."— Presentation transcript:

1 The Flu Network Astrid.Ferlinz@lifetech.com SMI Stockholm Steve Glavas Picture ISS Rome Gabriele Vaccari Picture CDC Atlanta Catherine Smith Picture JCVI Rockville David Wentworth Picture USDA – NVSL Ames, IA Mary Lea Killian Picture 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 Goals & Objectives: Evaluate & define protocol for Flu Typing Share & discuss results Make protocol/publication publicly available

2 RNA virus Genome size: approx 13.5 kb Comprised of 8 segments that encode up to 11 proteins Influenza A Virus 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 Rapid Influenza A virus typing on the Ion PGM TM Sequencer

3 PathAmp TM FluA Pre-Amplification Reagents PCR primerRT/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 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

4 Ion Torrent™Current CE (Sanger) Throughput10 samples/run (Ion 314™ chip) 1 sample/run DataWhole genomeH and N genes only TAT18 hrs/10 samples2-3 Days UseRUO, research, epidemiology, monitoring Screening (subset of samples only) SpecificitySub-types, mixed infectionsMain H/N variants only; single infections Cost115 Euro/sample*>€200 (H/N genes only) Ion Torrent™ vs current workflow Ion PGM™ Sequencing allows: Deeper understanding of genetic landscape and re-assortments within the Influenza A genome

5 Workflow 5 Reverse Transcription 20 µl with up to 8 µl Sample 60 min PCR 50 µl with ALL RT product 3 hrs Optional: Agarose gel analysis 8 segments from 2400 to 900 bp size Amplicon clean up & Quantitation DNA library prep Ion Xpress™ Plus Fragment Library Kit Enrichment of library Ion PGM™ 200 OneTouch™ Template Kit Sequencing Ion 314™/316™ Chip 30 min ~6 hrs5 hrs3 hrs MagMax™ Viral RNA Isolation Kit Nanodrop RNA Extraction MagMax™-96 Viral RNA Isolation Kit

6 Results & Comparison with CE Sequencing 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 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 Data provided by S Glavas, SMI, Stockholm Average Coverage Decrease of segment lengths Ion 316™ Chip

7 7 Application note Released April 2013 Collaborator & internal R&D data Application note Released April 2013 Collaborator & internal R&D data

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

9 PathAmp TM 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 PathAmp TM 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 Sequencing of the H7N9 virus during China Outbreak Flu Season Winter/Spring 2013

10 For Research Use Only. Not for use in diagnostic procedures. © 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. Start sequencing now at lifetechnologies.com/iontorrent Thank you !

11 © 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 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. Limitations and Disclaimers


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