1. 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
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JCVI Rockville David Wentworth
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ISS Rome
Gabriele Vaccari
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CDC Atlanta
Catherine Smith
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Goals & Objectives:
Evaluate & define protocol for Flu Typing
Share & discuss results
Make protocol/publication publicly available
USDA – NVSL Ames, IA
Mary Lea Killian
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2. 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:

3. 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

4. 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

5. 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

6. 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

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

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

9. 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

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

11. 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