Bioinformatics tools for - University of Connecticut
Bioinformatics tools for viral quasispecies reconstruction from next-generation sequencing data and vaccine optimization PD: Ion Mndoiu, UConn Co-PDs: Mazhar Khan, UConn Rachel ONeill, UConn Alex Zelikovsky, GSU Outline Background & aims of the project Bioinformatics tools for quasispecies spectrum
reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work Infectious Bronchitis Virus (IBV) Group 3 coronavirus Biggest single cause of economic loss in US poultry farms Young chickens: coughing, tracheal rales, dyspnea Broiler chickens: reduced growth rate Layers: egg production drops 5-50%, thin-shelled, watery albumin
IBV-infected egg defects Worldwide distribution, with dozens of serotypes in circulation Co-infection with multiple serotypes is not uncommon, creating conditions for IBV-infected recombination embryo normal
embryo IBV Vaccination Broadly used, most commonly with 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] RNA Virus Replication High mutation rate (~10-4) Lauring & Andino, PLoS Pathogens 2011 Evolution of IBV Quasispecies identified by cloning and Sanger sequencing in both IBV infected poultry and
commercial vaccines [Jackwood, Hilt, and Callison 2003; Hilt, Jackwood, and McKinley 2008] How Are Quasispecies Contributing to Virus Persistence and Evolution? Variants differ in
Virulence Ability to escape immune response Resistance to antiviral therapies Tissue tropism Lauring & Andino, PLoS Pathogens 2011 Project Aims Develop bioinformatics tools for accurate reconstruction of quasispecies sequences and their frequencies from next-generation reads Study quasispecies persistence and evolution of IBV in commercial layer flocks following
vaccination Use results of this study to optimize vaccine development and vaccination protocols Outline Background & aims of the project Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work Next Generation Sequencing
Illumina HiSeq 2000 Roche/454 FLX Titanium up to 6 billion PE reads/run 400-600 million reads/run 35-100bp read length Length up to 1,000 bp http://www.economist.com/node/16349358 10 Ion Torrent PGM 1-10M reads/run length up to 400bp
SOLiD 4/5500 1.4-2.4 billion PE reads/run 35-50bp read length Shotgun vs. Amplicon Reads Shotgun reads starting positions distributed ~uniformly Amplicon reads reads have predefined start/end positions covering fixed overlapping windows
Reconstruction from Shotgun Reads: ViSpA Shotgun reads Quasispecies sequences w/ frequencies Read Error Correction Frequency Estimation
Read Alignment Contig Assembly Preprocessing of Aligned Reads Read Graph Construction User Specified Parameters: (A) Number of mismatches
(B) Mutation rate Reconstruction from Amplicon Reads: VirA Errorcorrected SAM/BAM Read data Estimate Amplicons Amplicon Read Graph Reference in FASTA
format Viral population variants with frequencies Frequency Estimation Max-Bandwidth Paths Amplicon Sequencing Challenges Multiple reads from consecutive amplicons may match
over their overlap Distinct quasispecies may be indistinguishable in an amplicon interval Outline Background & aims of the project Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work IBV Genome
Rev. Bras. Cienc. Avic. vol.12 no.2 Campinas Apr./June 2010 RT-PCR of S1 using redesigned primers Experiment 1 M42 Sample 53 plasmid clones 10 clone pool
48945 M42 clone pool 21040 21040 19439 17122 Reads Validation
How well we predicted sanger clones How well our prediction is Average Prediction Error Neighbor-Joining Tree for M42 Sanger Clones & Vispa Qsps Experiment 2
Reads Statistics & Coverage Number of Reads Sample 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 Neighbor-Joining Tree for Sanger clones and ViSpA Reconstructed Sequences Outline Background & aims of the project Bioinformatics tools for quasispecies spectrum
reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work Summary Developed software tools for quasispecies reconstruction from both shotgun and amplicon next-generation reads Code and executables freely available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html http://alan.cs.gsu.edu/vira/ ViSpA plugin developed for users of ION Torrent, available on ION community
Experimental results on both simulated and real data show improved accuracy tradeoffs compared to previous methods Tools are applicable to quasispecies studies of other viruses Ongoing Work Deployment of ViSpA and VirA on Galaxy servers maintained at UConn and GSU Tool validation on ION Torrent reads Comparison of shotgun and amplicon based reconstruction methods Combining long and short read technologies Quasispecies persistence studies using longitudinal sampling Tool Validation for ION Torrent reads
Shotgun IBV reads generated using 316 ION chip 2,384,007 reads (1,177,740 after SAET correction) mean length 203.58 bp ViSpA results 23 quasispecies with estimated frequency > .5%, 2,200 total Longitudinal Sampling Amplicon / shotgun sequencing
Contributors Bassam Tork Ekaterina Nenastyeva Alex Artyomenko Serghei Mangul Nicholas Mancuso Alexander Zelikovsky University of Maryland Irina Astrovskaya, Ph.D. University of Connecticut: Rachel ONeal, PhD.
Mazhar Kahn, Ph.D. Hongjun Wang, Ph.D. Craig Obergfell Andrew Bligh
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