a phylogeny-based taxonomic characterization of the human
TRANSCRIPT
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A phylogeny-based taxonomic characterization of the human gut microbiome from shotgun
sequence dataAnalysis of HMP 454 WGS data with PhylOTU
Rebecca M. Lamb
University of California, San Francisco
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HMP 454 WGS data
• Whole Genome Sequencing (WGS)
• Gut samples
• 14 individuals
• 37M sequences
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PhylOTU Update
• Code more efficient and parallelized since publication
• Latest version available on github.com
• 24 hours of run time (60 parallel jobs)
• 59K 16S reads classified into OTUs
• 1775 OTUs @ equivalent 97% PID
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Silva reference tree
1 representative from each gut genus
ActinobacteriaBacteroidetes
Proteobacteria
Firmicutes
VerrucomicrobiaLentisphaerae
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Actinobacteria
Bacteroidetes
Proteobacteria
Firmicutes
VerrucomicrobiaLentisphaerae
Number of OTUsRDP classification
• 0.5 bootstrap cutoff for each read
• >50% sequences in OTU agree
• 40% of OTUs unidentified
198 OTUs
10 OTUs
Log scale:
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Numbers of novel OTUs
Definition Full length typed or identified
RDP DB
Full RDP DB
Novel OTUs No RDP match @ 95% coverage, 95% IDto any read in the OTU
410 49
Novel + Illumina Support
Illumina read match to 454 read80bp @ 99% ID, 0 gaps
230 23
Novel + Core
454 or Illumina reads present in ≥7 (50%) individuals
37 2
• 3.5M 16S reads from Illumina WGS of 12 of the same individuals
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs
1 OTU
106 OTUs
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs
1 OTU
106 OTUs
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Novel OTUs are distributed
across the tree
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs (no support)
1 OTU
106 OTUs
Novel + Illumina support
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs (no support)
1 OTU
106 OTUs
Novel + Illumina support
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
High confidence novel OTUs are
distributed across the tree
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs (no support)
1 OTU
106 OTUs
Novel + Illumina support
Novel + Core
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
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Novel OTUscompared to RDP full length typed or identified sequences
Novel OTUs (no support)
1 OTU
106 OTUs
Novel + Illumina support
Novel + Core
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Some core OTUs are very poorly
identified
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Novel OTUs
1 OTU
13 OTUs
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Novel OTUscompared to full RDP database
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Novel OTUs
1 OTU
13 OTUs
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Novel OTUscompared to full RDP database
Strictly defined novel OTUs are
relatively phylogenetically
restricted
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13 OTUs
Novel OTUs (no support)
1 OTU
Novel + Illumina support
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Novel OTUscompared to full RDP database
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13 OTUs
Novel OTUs (no support)
1 OTU
Novel + Illumina support
Actinobacteria Bacteroidetes
Proteobacteria
Firmicutes
Novel OTUscompared to full RDP database
High confidence novel OTUs in
Firmicutes, Proteobacteria, Bacteroidetes
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Firmicutes – Clostridia - Clostridiales – Ruminococcaceae – Anaerotruncus
Proteobacteria – Betaproteobacteria – Burkholderiales – Alcaligenaceae – Parasutterella
Novel OTUscompared to full RDP database
• 1 454 Read, 1.0 RDP bootstrap• 243 Illumina reads from 7 individuals• Parasutterella (2009)
• 21 other OTUs identified as this genus• Gut associated
• 1 454 Read, 0.65 RDP bootstrap • 20 Illumina reads from 7 individuals• Anaerotruncus (2004)
• 21 other OTUs identified as this genus• Gut associated• Associated with bacteraemia
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Conclusions
• Identified gut microbiome diversity below the genus level
• Novel OTUs identified– ~50% supported with Illumina reads
– ~9% are present in multiple individuals
• Come visit my poster
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Acknowledgements
Makedonka MitrevaGeorge WeinstockErica SodergrenHongyu Gao
Katherine S. PollardThomas J. Sharpton
Anthony Fodor
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Backup
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RDP classification• 0.5 bootstrap cutoff for each read
• >50% sequences in OTU agree
• 40% of OTUs unidentified
(inner ring)
Actinobacteria
Bacteroidetes
Proteobacteria
Firmicutes
VerrucomicrobiaLentisphaerae
Number of OTUs
198 OTUs
10 OTUs
Log scale:
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Actinobacteria
Bacteroidetes
Proteobacteria
Firmicutes
VerrucomicrobiaLentisphaerae