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