The Flu [email protected]
SMI StockholmSteve Glavas
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ISS RomeGabriele Vaccari
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CDC AtlantaCatherine Smith
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JCVI Rockville David Wentworth
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USDA – NVSL Ames, IAMary Lea Killian
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SMI Smittskyddsinstitutet ISS Istituto Superiore di SanitàCDC Centers of Disease ControlJCVI J Craig Venter InstituteUSDA – NVSL US Dep of Agriculture – National Veterinary Service Labs
Goals & Objectives:Evaluate & define protocol
for Flu TypingShare & discuss results
Make protocol/publication publicly available
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 PGMTM Sequencer
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™ 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)
Ion Torrent™ vs current workflow
Ion PGM™ Sequencing allows:Deeper understanding of genetic landscape and re-assortments within the Influenza A genome
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 hrs 5 hrs 3 hrs
MagMax™ Viral RNA Isolation KitNanodrop
RNA Extraction
MagMax™-96 Viral RNA Isolation Kit
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
Data provided by S Glavas, SMI, Stockholm
Ave
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Decrease of segment lengths
Ion 316™ Chip
PathAmpTM FluA Pre-Amplification Reagents were used to sequence whole-genome of H7N9Highly accurate and sensitive results from both swab samples and isolated virus samplesDetection of mixed infectionsPublication submitted
Sequencing of theH7N9 virus during China Outbreak
Flu Season Winter/Spring 2013
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 atlifetechnologies.com/iontorrent
Thank you !
© 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