Pathogen Profiling Pipeline

June 27, 2009 Pathogen Profiling PipelineM3 SIG – ISMB/ECCB 2009

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Pathogen Profiling Pipeline

Tom MatthewsNational Microbiology LaboratoryPublic Health Agency of Canada

[email protected]

A Metagenomics Tool for RapidIdentification of Pathogens from Clinical

Specimens


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Introduction

● With novel/emerging disease classical pathogen identification may not always produce results

● Advances in next-gen sequencing technology● Characterize samples at genomic level

● Pathogen Profiling Pipeline● Bioinformatics pipeline ● Analysis of host and microbial nucleic acids


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Features

● Nucleotide and protein BLAST analysis● Unbiased analysis of input reads● Clustered execution● Web front-end● Custom analysis pipelines● Easily viewed results


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Filtering Overview● BLAST analysis performed against reference

sequence database● Assigns hits according to cut-off criteria● Calculate equivalent hits● Clustered BLAST and filtering


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Last Common Ancestor Estimation

● Uses equivalent hits for LCA calculation

● User specifies equivalent hit percentage cutoff

● NCBI taxonomy database for ancestor lookup

● Walks up taxonomy tree to find lowest intersection of all leaf nodes

● Unbiased approach

Vaccinia

Camelpox

Taterapox

VariolaOrthopoxvirus


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Filtering Outputs

● Hits – High scoring reads passing filtering values

● Equivalent Hits – BLAST hits matching to within an assigned percentage of the top hit's bitscore

● Last Common Ancestors – Calculated (estimated) LCA of all the equivalent hits

● Unassigned – Passed to the next pipeline step


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Example Analysis Method

● BLAST reads against host database

● Remove host reads

● BLAST unassigned against reference database

● Filter hits vs. unassigned

● Repeat...

● Post analysis

Samplereads

BLAST andFiltering

Hostgenome

Viralgenome

Bacterialgenome

Protozoangenome

Fungalgenome

Non-hostreads

BLAST andFiltering

BLAST andFiltering

Non-hostreads

BLAST andFiltering

BLAST andFiltering

Poolresults

UniqueorganismsIn sample


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Pipeline Construction


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Pipeline Execution

● Custom execution manager● Computes dependencies and monitors running

jobs● Distribute jobs across Linux cluster● Facilitates unattended clustered executions


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Reports


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Drill Down Reports


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Abundance View

● Displays abundance of taxonomic hits


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Example Run

● Mouth swab input samples● Two pools:

● Samples spiked with Vaccinia and Influenza A● Background reference sample


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Example Run


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Example run


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Example Run


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Example Run


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Wrap-up

● Unbiased analysis of input reads● Custom analysis pipelines● Last common ancestor calculation● Clustered execution● Multiple report views● Exportable results


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Acknowledgements

● Gary Van Domselaar● Morag Graham● Shaun Tyler● Heather Kent● Kim Melnychuk● Christine Bonner● Geoff Peters● Philip Mabon

Pathogen Profiling Pipeline

Technology