Download presentation
Published byArabella Benson Modified over 9 years ago
1
Bioinformatics tools for viral quasispecies reconstruction from next-generation sequencing data and vaccine optimization PD: Ion Măndoiu, UConn Co-PDs: Mazhar Khan, UConn Rachel O’Neill, UConn Alex Zelikovsky, GSU
2
Outline Background & aims of the project
Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work
3
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 Worldwide distribution, with dozens of serotypes in circulation Co-infection with multiple serotypes is not uncommon, creating conditions for recombination IBV-infected egg defects IBV-infected embryo normal embryo
4
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]
5
Lauring & Andino, PLoS Pathogens 2011
RNA Virus Replication High mutation rate (~10-4) Lauring & Andino, PLoS Pathogens 2011
6
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]
7
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
8
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
9
Outline Background & aims of the project
Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work
10
Next Generation Sequencing
Illumina HiSeq 2000 up to 6 billion PE reads/run 35-100bp read length Roche/454 FLX Titanium million reads/run Length up to 1,000 bp Ion Torrent PGM 1-10M reads/run length up to 400bp SOLiD 4/5500 billion PE reads/run 35-50bp read length 10
11
Shotgun vs. Amplicon Reads
1111 Shotgun vs. Amplicon Reads Shotgun reads starting positions distributed ~uniformly Amplicon reads reads have predefined start/end positions covering fixed overlapping windows
12
Reconstruction from Shotgun Reads: ViSpA
Error Correction Read Alignment Preprocessing of Aligned Reads Read Graph Construction Contig Assembly Frequency Estimation Shotgun reads Quasispecies sequences w/ frequencies User Specified Parameters: (A) Number of mismatches (B) Mutation rate
13
Reconstruction from Amplicon Reads: VirA
Error-corrected SAM/BAM Read data Estimate Amplicons Amplicon Read Graph Reference in FASTA format Viral population variants with frequencies Frequency Estimation Max-Bandwidth Paths
14
Amplicon Sequencing Challenges
1414 Amplicon Sequencing Challenges Multiple reads from consecutive amplicons may match over their overlap Distinct quasispecies may be indistinguishable in an amplicon interval
15
Outline Background & aims of the project
Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work
16
IBV Genome RT-PCR of S1 using redesigned primers
Rev. Bras. Cienc. Avic. vol.12 no.2 Campinas Apr./June 2010 RT-PCR of S1 using redesigned primers
17
Assembled quasispecies
Experiment 1 10 clone pool C1 20% C2 20% C3 15% C4 15% C5 10% C6 10% C7 4% C8 4% C9 1% C10 1% Assembled quasispecies PV1 PV2 PV3 … PVk 454 reads M42 Sample 53 plasmid clones V1 V2 V3 Vn
18
Evaluated Reconstruction Flows
19
Reads Statistics & Coverage
Sample Number of Reads Uncorrected SAET Corrected Shorah Corrected KEC Corrected M42 isolate 53062 50858 48945 M42 clone pool 21040 19439 17122
20
Reads Validation
21
How well we predicted sanger clones
How well our prediction is
22
Average Prediction Error
23
Neighbor-Joining Tree for M42 Sanger Clones & Vispa Qsps
24
Experiment 2
25
Reads Statistics & Coverage
Sample Number of Reads Uncorrected SAET corrected Shorah corrected KEC corrected M41 Vaccine 92113 87883 85311 Field #1 38502 33685 32521 Field #2 132513 123370 111686 Field #3 76906 71408 64507 Field #4 44467 41653 37295
26
Neighbor-Joining Tree for Sanger clones and ViSpA Reconstructed Sequences
27
Outline Background & aims of the project
Bioinformatics tools for quasispecies spectrum reconstruction from NGS reads Experimental validation on IBV data Summary and ongoing work
28
Summary Developed software tools for quasispecies reconstruction from both shotgun and amplicon next-generation reads Code and executables freely available at 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
29
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
30
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 bp ViSpA results 23 quasispecies with estimated frequency > .5%, 2,200 total
31
Longitudinal Sampling
Amplicon / shotgun sequencing
32
University of Connecticut:
Contributors Bassam Tork Ekaterina Nenastyeva Alex Artyomenko Serghei Mangul Nicholas Mancuso Alexander Zelikovsky University of Connecticut: Rachel O’Neal, PhD. Mazhar Kahn, Ph.D. Hongjun Wang, Ph.D. Craig Obergfell Andrew Bligh University of Maryland Irina Astrovskaya, Ph.D.
Similar presentations
© 2024 SlidePlayer.com. Inc.
All rights reserved.