Pathogen Profiling Pipeline
A Metagenomics Tool for Rapid
Identification of Pathogens from Clinical
Specimens
Tom Matthews
National Microbiology Laboratory
Public Health Agency of Canada
thomas_matthews@phac-aspc.gc.ca
June 27, 2009 Pathogen Profiling Pipeline 1
M3 SIG – ISMB/ECCB 2009

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
Variola
Orthopoxvirus
Taterapox
Camelpox
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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
Sample
reads
● BLAST reads against host
database Host
genome
BLAST and
Filtering
● Remove host reads Non-host
reads
● BLAST unassigned against
reference database Viral BLAST and
Filtering
genome
● Filter hits vs. unassigned Bacterial BLAST and
genome Filtering
● Repeat... Pool
results
Protozoan BLAST and
● Post analysis genome Filtering
Unique
Fungal BLAST and
organisms
genome Filtering
In sample
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Pipeline Construction
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Pipeline Construction
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Pipeline Construction
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Pipeline Construction
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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
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