Bassam Tork Georgia State University Atlanta, GA 30303, USA
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Transcript of Bassam Tork Georgia State University Atlanta, GA 30303, USA
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Bioinformatics Methods for Reconstruction of Infectious Bronchitis Virus Quasispecies from
Next Generation Sequencing Data
Bassam Tork
Georgia State UniversityAtlanta, GA 30303, USA
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2
Outline
• Introduction• Reconstruction of Quasispecies from Shotgun
Reads.• Experiment Results• Future Work.
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Infectious Bronchitis Virus (IBV) Group 3 coronavirus Economic loss in US poultry farms
– Young chickens– Broiler chickens– Layers
Worldwide distribution, with dozens of serotypes in circulation• Co-infection with multiple serotypes is not
uncommon, creating conditions for recombination
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healthy chicks
IBV-infectedembryo
normalembryo
IBV-infectedegg defect
Infectious Bronchitis Virus (IBV)• Main cause of economic loss in US poultry farms
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• Broadly used; attenuated live vaccine- Short lived protection- Layers need to be re-vaccinated multiple times during their
lifespan- Vaccines might undergo selection in vivo and regain
virulence [Hilt, Jackwood, and McKinley 2008]
IBV Vaccination
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How Are Quasispecies Contributing to Virus Persistence and Evolution?
• Variants differ in– Virulence– escape immune response– Resistance to antiviral therapies
Lauring & Andino, PLoS Pathogens 2011
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Next Generation Sequencing and
IBV Develop computational methods to study
quasispecies evolution pre and post vaccination
Optimize vaccination Strategies
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+ Ion TorrentIon Proton
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Evolution of IBV
Taken from Rev. Bras. Cienc. Avic. vol.12 no.2 Campinas Apr./June 2010
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ViSpA:Viral Spectrum Assembler
User Specified Parameters: (A) Number of mismatches (B) Mutation rate
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Experiment1A B
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Reads Statistics & Coverage
Sample
Number of Reads
Uncorrected SAET Corrected Shorah Corrected KEC Corrected
M42 isolate 53062 53062 50858 48945
M42 clone pool 21040 21040 19439 17122
0 200 400 600 800 1000 1200 1400 1600 1800 20000
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M42
Position in S1 Gene
Read
Cov
erag
e
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Reconstructed Quasispecies Variability
*IonSample42RL1.fas_KEC_corrected_I_2_20_CNTGS_DIST0_EM20.txt
Sequencing primer ATGGTTTGTGGTTTAATTCACTTTC
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Pairwise Edit Distance between 10 Clone Pool
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
0 0 2 2 1 3 4 42 7 3 C1
0 2 2 1 2 3 41 6 2 C2
0 4 3 5 6 44 9 5 C3
0 1 5 4 42 9 3 C4
0 4 3 41 8 2 C5
0 6 40 6 4 C6
0 45 11 4 C7
0 41 41 C8
0 8 C9
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Quasispecies Reconstruction Flows
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Reads Validation
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How well we predicted sanger
clones
How well our prediction is
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Average Prediction Error
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A: M42 Sanger & ViSpA NJ Tree
B: M42 10 Clone Sangers & ViSpA NJ Tree
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Experiment2
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Reads Statistics & CoverageSample
Number of Reads
Uncorrected SAET corrected Shorah corrected KEC corrected
M41 Vaccine 92113 92113 87883 85311
Field #1 38502 38502 33685 32521
Field #2 132513 132513 123370 111686
Field #3 76906 76906 71408 64507
Field #4 44467 44467 41653 37295
0 200 400 600 800 1000 1200 1400 1600 1800 20000
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M41 Vaccine
Position in S1 Gene
Read
Cov
erag
e
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Vaccine Sanger & ViSpA NJ Tree
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Future Work
• Comparison of shotgun and amplicon based reconstruction methods
• Quasispecies reconstruction from Ion Torrent reads
• Combining long and short read technologies• Optimization of vaccination strategies
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Contributors
University of Connecticut:Rachel O’Neal, PhD.
Ion Mandiou, PhD.Mazhar Kahn, Ph.D.
Hongjun Wang, Ph.D. Craig ObergfellAndrew Bligh
Bassam TorkEkaterina Nenastyeva
Alex ArtyomenkoSerghei Mangul
Nicholas MancusoAlexander Zelikovsky
University of MarylandIrina Astrovskaya, Ph.D.
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Fundings
University of Connecticut:
Molecular Basis of Disease Program
Georgia State
University:
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Thanks