The Flu NetworkAstrid.Ferlinz@lifetech.com

SMI StockholmSteve Glavas

Picture

ISS RomeGabriele Vaccari

Picture

CDC AtlantaCatherine Smith

Picture

JCVI Rockville David Wentworth

Picture

USDA – NVSL Ames, IAMary Lea Killian

Picture

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

rag

e C

ove

rag

e

Decrease of segment lengths

Ion 316™ Chip

7

Application note Released April 2013

Collaborator & internal R&D data

Data analysis plugin Launched in IC in April 2013

by collaborators from SMI

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