NGS, omics and applied bioinformatics at CVI CVI Bioinformatics practise, tips and tricks…
Alex Bossers, Freddy de Bree, and Frank Harders et al.
Department of Infection Biology
PathogenOmics group
Nbic::Bioassist 16 September 2011
www.cvi.wur.nl
CVI - Lelystad
Part of WUR – Animal Sciences Group CVI ~250 employees Co-location ASG-Lifestock Research
www.cvi.wur.nl
CVI - Lelystad
Animal disease control Prevention of transmission to humans
Diagnoses and crisis organization Development of animal models and methods/pathobiology Development of models (epidemiology) Development of diagnostic tests Development of intervention tools (vaccines, therapeutics) Pathobiology, animal models and clinical studies
www.cvi.wur.nl
CVI – Department of Infection Biology
Host-pathogen interactions OMICS:
• Proteome / transcriptome / metabolome Immunology and pathogenesis
Vaccine and diagnostics development
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bioinformatics at CVI Applied bioinformatics
CVI: Frank, Freddy, Alex Small base so collaborations are crucial
• Life stock Research (ASG) and Plant Research International (PRI) • AMC, Uni Manchester, Uni Maryland/NBACC, Roslin, Sanger, and others…
Current/past focus Reversed vaccinology approaches (sequence/knowledge-based vaccine design) NGS CGH/tiling array approaches (molecular epidemiology) In situ proteome array approaches Pathogen genome sequencing and reconstruction Transcriptomics and analysis HT genotyping and automation
Future focus (2012-)
Virusdiscovery and pathogen identification (NGS and DEFRA biochip :: EpiZone chip) NGS transcriptomics vs arrays Pathogen detection/typing solutions (arrays (solid/solution/tubes)) Metagenomics (NGS)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
NGS - CVI
CVI: Sanger capillary sequencing and array platforms
Roche 454
Sequencing unit AMC (Amsterdam)
Onderstepoort Veterinary Institute (South Africa)
Illumina at ServiceXS and BaseClear (Leiden)
November 2011: Illumina MiSeek
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Pathogens - focus
Coxiella burnetii (Q fever) Streptococcus suis
ESBLs in several species others...
Blue Tongue (BTV) Avian influenza AngHV1 (eel) Prions/TSEs others…
Knowledge-based vaccine design
Reversed vaccinology approaches Sequence (knowledge) based vaccine design Vaccine-candidate reduction
• In silico
– Localization – Motifs for post-translational modifications and immunomodulation
• In vitro by proteome approaches – 2D, SELDI/mass-spec, Western blots,… – In situ protein/proteome arrays
Knowledge building host::pathogen interactions • Transcriptome of host and pathogen
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Reversed vaccinology
Genome 2-4Mb
Proteome 2-4k ORFs
In silico
approaches
2D
SELDI
Localization
Predict exposed antigens
Motifs for modification (lipo)
In situ protein arrays
Candidate reduction for vaccines/diagnostics
Genome variation/plasticity
ReversedReversed
Genome4Mb
Reversed
Genome2-4Mb
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
NGS analysis flow
Raw NGS data 454 shotgun, PE 3kb
Illumina mp50, PE100
BowtieMapping
Filter data
FastQC
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bowtie mapping to check/filter contaminants
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
NGS analysis flow
Raw NGS data 454 shotgun, PE 3kb
Illumina mp50, PE100
BowtieMapping
Gap closure
Filter data
de novo assembly
Scaffolding contigs
FastQC
Newbler (454)
MIRA (mixed)
ABYSS (mixed)
MUMmer / BLA(S)T (reference)
SSPACE (PE data)
IMAGE (PE data)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
NGS analysis flow
Raw NGS data 454 shotgun, PE 3kb
Illumina mp50, PE100
BowtieMapping
Gap closure
Filter data
de novo assembly
Scaffolding contigs
FastQC
Newbler (454)
MIRA (mixed)
ABYSS (mixed)
MUMmer / BLA(S)T (reference)
SSPACE (PE data)
IMAGE (PE data) Comparisons (Mumer)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Genome comparisons (MUMmer)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Genome annotation (visualised Artemis)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
CVI (galaxy) servers
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Tool versioning
Subversion repositories Personal Galaxy
• Development server • Production server • Project specific galaxy modifications
Assembla issue/bug/feature tracking and ticketing system
Wiki for developers
Galaxy toolshed
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Tool versioning
Subversion repositories Personal Galaxy
• Development server • Production server • Project specific modified galaxy instance(s)
Assembla.com bug/feature tracking and ticketing system
Wiki for developers
Galaxy toolshed
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Software tools Notes
Evernote ~ electronic journal (free win/mac/android/linux) Doit.im for Getting Things Done (free all major platfoms) Freemind mindmapping ManicTime (time tracking)
Version/source control Subversion repositories and ticketing at Assembla Eclipse (IDE for perl, MySQL, R, some php) PyCharm / PHPStorm (python/PHP IDE from jetbrains) dbWrench (DB administration)
Linux command line Bash / sed / grep
Others R Galaxy
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Evern
ote
exam
ple
(Lin
ux N
evern
ote
)
Favourite websites
Galaxy Tool syntax http://wiki.g2.bx.psu.edu/Admin/Tools/Tool%20Config%20Syntax
PerlMonks
BioStar.stackoverflow.com (ALchEmiXt)
SeqAnswers.com
CodeIgniter PHP framework (MVC)
Assembla.com
Ubuntu server guide
Geek and Poke (http://geekandpoke.typepad.com)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
Challenges and upcoming work
Work Only very limited datasets yet Complete several genomes for publication Improve reversed vaccinology tools
• Annotations/motifs • Easy filtering differences
Link galaxy directly to Artemis and ACT Challenges
Let agent expert do the work Virusdiscovery NGS transcriptomics (RNAseq)
STORAGE!
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011
© Wageningen UR
Questions…
[email protected] (twitter / BioStar / SeqAnswers)
Bossers et al. NBIC-BioAssist meeting, Sep 16th 2011