Ion PGM™ Sequencing allows

download report

Transcript Ion PGM™ Sequencing allows

The Flu Network [email protected]

JCVI Rockville David Wentworth Picture SMI Stockholm Steve Glavas Picture

SMI ISS

Smittskyddsinstitutet Istituto Superiore di Sanità

CDC

Centers of Disease Control

JCVI

J Craig Venter Institute

USDA – NVSL

US Dep of Agriculture – National Veterinary Service Labs ISS Rome Gabriele Vaccari Picture USDA – NVSL Ames, IA Mary Lea Killian Picture CDC Atlanta Catherine Smith Picture

Goals & Objectives:

Evaluate & define protocol for Flu Typing Share & discuss results Make protocol/publication publicly available

Rapid Influenza A virus typing on the Ion PGM

TM

Sequencer Influenza A Virus

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 1: major component of virion Matrix protein 2: Integral membrane protein – Ion Channel Non-structural protein 1: RNA transport, translation, splicing Non-structural protein 2: function not known

PathAmp

TM

FluA Pre-Amplification Reagents

Core consensus 5’ Core consensus 3’ PCR primer RT/PCR primer Influenza A virus genomic RNAs

•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

Throughput Data TAT

Ion Torrent™

10 samples/run (Ion 314™ chip) Whole genome 18 hrs/10 samples

Current CE (Sanger)

1 sample/run H and N genes only 2-3 Days Use RUO, research, epidemiology, monitoring Screening (subset of samples only) Specificity Cost Sub-types, mixed infections 115 Euro/sample* Main H/N variants only; single infections > €200 (H/N genes only)

Ion PGM™ Sequencing allows:

Deeper understanding of genetic landscape and re-assortments within the Influenza A genome

Workflow

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

Results & Comparison with CE Sequencing

Ion 316™ Chip Data provided by S Glavas, SMI, Stockholm Decrease of segment lengths 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

Application note

Released April 2013

Collaborator & internal R&D data

Data analysis plugin

Launched in IC in April 2013 by collaborators from SMI

Sequencing of the H7N9 virus during China Outbreak

Flu Season Winter/Spring 2013

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

Thank you !

Start sequencing now at lifetechnologies.com/iontorrent

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.

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